Food and Chemical Toxicology 43 (2005) 985–1015

www.elsevier.com/locate/foodchemtox

Review

Safety assessment of esters of p-hydroxybenzoic acid (parabens)

a,*
M.G. Soni , I.G. Carabin b, G.A. Burdock c

a
Burdock Group, 2001 9th Avenue, Suite 3001, Vero Beach, FL 32960, USA
b
Women’s Health Science Institute Inc., Vero Beach, FL 32968, USA
c
Burdock Group, 888 17th St. N.W., Suite 810, Washington, DC 20006, USA

Received 23 December 2004; accepted 31 January 2005

Abstract

Parabens are widely used as preservatives in food, cosmetic and pharmaceutical products. Acute, subchronic, and chronic studies
in rodents indicate that parabens are practically non-toxic. Parabens are rapidly absorbed, metabolized, and excreted. In individuals
with normal skin, parabens are, for the most part, non-irritating and non-sensitizing. However, application of compounds contain-
ing parabens to damaged or broken skin has resulted in sensitization. Genotoxicity testing of parabens in a variety of in vitro and in
vivo studies primarily gave negative results. The paraben structure is not indicative of carcinogenic potential, and experimental stud-
ies support these observations. Some animal studies have reported adverse reproductive effects of parabens. In an uterotrophic
assay, methyl and butyl paraben administered orally to immature rats were inactive, while subcutaneous administration of butyl
paraben produced a weak positive response. The ability of parabens to transactivate the estrogen receptor in vitro increases with
alkyl group size. The detection of parabens in a small number of breast tumor tissue samples and adverse reproductive effects of
parabens in animals has provoked controversy over the continued use of these substances. However, the possible estrogenic hazard
of parabens on the basis of the available studies is equivocal, and fails to consider the metabolism and elimination rates of parabens,
which are dose, route, and species dependent. In light of the recent controversy over the estrogenic potential of parabens, conduct of
a reproductive toxicity study may be warranted.

2005 Elsevier Ltd. All rights reserved.

Keywords: Parabens; Preservative; Antimicrobial; Food ingredient; Food additive; Cosmetic; Drug; Excipient; Safety

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................. 986

1.1. Description, properties and sources. . . . . . . . . . . . . . . . . . . . . .................................. 986
1.2. Economic uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................................. 987
1.2.1. Uses as food ingredient . . . . . . . . . . . . . . . . . . . . . . ................................. 987
1.2.2. Uses in cosmetics . . . . . . . . . . . . . . . . . . . . . . . . . . ................................. 987
1.2.3. Pharmaceutical uses . . . . . . . . . . . . . . . . . . . . . . . . ................................. 988
1.2.4. Regulatory history. . . . . . . . . . . . . . . . . . . . . . . . . . ................................. 988

Abbreviations: DNCB, dinitrochlorobenzene; FEMA, flavor and extract manufacturersÕ association; GRAS, generally recognized as safe; MIC,
minimum inhibitory concentration; NOAEL, no observed adverse effect level; NOEL, no observed effect level; OTC, over the counter; PGST,
placental glutathione S-transferase; RIPT, repeated insult patch test; SCOGS, Select Committee on GRAS Substances.
*
Corresponding author. Tel.: +1 772 562 3900; fax: +1 772 562 3908.
E-mail address: msoni@burdockgroup.com (M.G. Soni).

0278-6915/$ - see front matter
2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2005.01.020
986 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015

1.3. Consumption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 989

1.3.1. Exposure to parabens via food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 989
1.3.2. Exposure to paraben via cosmetics and drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 990
1.3.3. Consumption summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 990
2. Biological data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 990
2.1. Absorption, metabolism and excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 990
2.1.1. Biochemical/pharmacological effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 993
2.2. Toxicological studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 993
2.2.1. Acute toxicity studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 993
2.2.2. Irritation studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 994
2.2.3. Sensitization studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 994
2.2.4. Short-term and subchronic toxicity studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 995
2.2.5. Chronic toxicity/carcinogenicity studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 996
2.2.6. Teratogenicity/reproduction toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 998
2.2.7. Genotoxicity studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 999
2.2.8. Cytotoxicity studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000
2.2.9. Estrogenic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1001
2.2.10. Neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1003
2.2.11. Antimicrobial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1003
2.3. Observations in humans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1004
2.3.1. Clinical observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1004
2.3.2. Human irritation and sensitization studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1005
2.3.3. Photocontact sensitization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1005
2.3.4. Case reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1005
2.3.5. Paraben paradox . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1006
2.3.6. Estrogenic activity in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1007
3. Risk evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1008
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1010

1. Introduction Several independent animal and human studies on

Parabens are a group homologous series of hydroxy-
benzoic acid, esterified at the C-4 position (including
methyl-, ethyl-, propyl-, butyl-, heptyl- and benzyl-para-
ben), used singly or in combination to exert an anti-
microbial effect. Parabens can have multiple biological
actions, but it is generally believed that their inhibitory
effects on membrane transport and mitochondrial func-
tion processes are key for their actions. Parabens were
first used as antimicrobial preservatives in pharmaceuti-
cal products in the mid-1920s (Sabalitschka, 1930). Para-
bens have proved to be very effective antimicrobial
agents and are widely used. As the chain length of the
ester group of paraben increases, antimicrobial activity
increases, but water solubility decreases (Elder, 1984).
Generally, the microbial replication occurs in the water
phase and hence, the amount of paraben dissolved in
the water phase determines the preservative ability. As
a result, generally the lower esters (methyl and propyl)
are the practical choices for use of parabens in foods.
The parabens meet several of the criteria of an ideal
preservative, in that they possess a broad spectrum of
antimicrobial activity, are safe to use (i.e., relatively
non-irritating, non-sensitizing, and low toxicity), are sta-
ble over the pH range and are sufficiently soluble in water
to produce the effective concentration in aqueous phase.
the safety of parabens have appeared in the published
literature. Additionally, some review articles on the
safety of parabens based on the published reports have
also appeared. Recent reports have indicated that expo-
sure to parabens may modulate or disrupt the endocrine
system and thus may have harmful consequences on hu-
man health. These studies have indicated that parabens
possess weak estrogenic activity, while others have
hypothesized an association between the use of under-
arm cosmetics containing parabens and an increased
incidence of breast cancer. Additionally, some recent
studies have reported adverse reproductive effects of
parabens. In view of the recent controversy on the safe
use of parabens, the objective of the present review is
to critically evaluate the available information on the
safety of parabens from non-clinical and clinical studies,
along with adverse events from the clinical reports, to
determine the safety in-use of parabens.
1.1. Description, properties and sources
Parabens are esters of 4-hydroxybenzoic acid
(Fig. 1), only differing in the ester group, which may
be a methyl-, ethyl-, propyl- or butyl-group. These ester
groups result in the formation of methyl paraben (CAS
No. 99-76-3), ethyl paraben (CAS No. 120-47-8), propyl
 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015 987

O uses of the parabens in the food industry are cakes, pas-

HO C O
tries, pie-crusts, icings, toppings and fillings (0.03–0.06%
R
of a 3:1 in ratio methyl and propyl parabens); soft drinks
(0.03–0.05% of a 2:1 ratio of methyl and propyl para-
Fig. 1. Chemical structure of paraben, where R = methyl (CH3), ethyl bens); creams and pastes (0.1% of a combination of
(C2H5), propyl (C3H7), butyl (C4H8).
parabens); jams, jellies and preserves (0.07% of a 2:1
ratio of methyl and propyl parabens); olives and pickles
paraben (CAS No. 94-13-3), or butyl paraben (CAS No. (0.1% of a combination of parabens); and syrups
94-26-8). The most frequently used parabens are methyl- (0.07%). Among the parabens, methyl and propyl para-
and propyl paraben. In pure form, parabens are gener- bens are the most extensively used in foods. To deter-
ally small colorless crystals or crystalline powders with mine the specific foods in which parabens were used
virtually no odor or taste; they are hygroscopic in nature and at what levels, a representative cross-section of food
and have a high oil/water coefficient. Parabens are pre- manufacturers was surveyed (FDA, 1973). A 30-fold in-
pared by esterification of p-hydroxybenzoic acid with crease in use of parabens was noted from 1960 to 1970.
the corresponding alcohol in the presence of an acid cat-
alyst. Generally, parabens are stable in air and are resis- 1.2.2. Uses in cosmetics
tant to hydrolysis in water (hot and cold) and in acidic Parabens are routinely used as preservatives in cos-
solutions. An increase in alkyl chain length of parabens metics, with propyl- and methyl paraben being the most
increases the resistance to hydrolysis. Appreciable commonly used (Berke et al., 1982; Jackson, 1992).
hydrolysis occurs at pH above 7. Table 1 summarizes These parabens are used in nearly all types of cosmetics,
some physicochemical properties of parabens. with reported use in over 13,200 formulations (Elder,
Parabens occur naturally in foods. Methyl paraben 1984). Parabens formulate well because they have no
has been reported as a constituent of cloudberry, yellow perceptible odor or taste, they do not produce discolor-
passion fruit juice, white wine, botrytised wine, and ation, they are practically pH neutral and they do not
Bourbon vanilla, and recently, propyl paraben has been cause hardening or ‘‘muddying’’ (Neidig and Burrell,
detected in the aerial part of the plant Stocksia brahuica 1944). The popular use of paraben preservatives in cos-
(Family: Sapindaceae) (Ali et al., 1998). Several investi- metics and toiletries arises from their low toxicity, broad
gators reported methods for determination of para- spectrum of activity, inertness, worldwide regulatory
bens in cosmetics, food products and pharmaceuticals acceptance, biodegradability and low cost. An addi-
(Mahuzier et al., 1927; Wang and Chang, 1998; Ali tional advantage is their excellent chemical stability in
et al., 1999; Kreuz et al., 1999; Driouich et al., 2000; relation to pH (effective between pH 4.5–7.5) and tem-
Lin Yu et al., 2000; Labat et al., 2000). perature. Because of their stability at high temperature,
products containing parabens can be safely autoclaved
without significant loss of antimicrobial activity, as a
1.2. Economic uses
result of hydrolysis (Maddox, 1982).
Parabens individually, or in combination, are used in
1.2.1. Uses as food ingredient
all cosmetic product formulation categories. Products
Parabens have been added to food for more than
containing these preservatives may contact the skin,
50 years and, over the years, the use of parabens has
hair and scalp, lips, mucosae (oral, ocular, and vaginal),
steadily increased to include many more food categories.
axillae and nails. Doron et al. (2001) proposed use of
They are employed in several foods including processed
parabens, including propyl paraben, as potential anti-
vegetables, baked goods, fats and oils, seasonings, sugar
bacterial agents against immobilized or planktonic (free
substitutes, coffee extracts, fruit juices, pickles, sauces,
floating) bacteria found in the oral cavity. The products
soft drinks and frozen dairy products at concentrations
containing parabens may be used on an occasional or a
of between 450 and 2000 ppm (Daniel, 1986). The major
consistent basis and their use may extend over a period
of years. In some instances, frequency and duration of
application may be continuous. Use of paraben, alone
Table 1
General description and physicochemical properties of parabens or in combination with other compounds, is well suited
for the preservation of cosmetics (Maddox, 1982).
Characteristics Methyl Ethyl Propyl Butyl
Parabens are most active against molds and yeasts,
CAS no. 99-76-3 120-47-8 94-13-3 94-26-8
and have been successfully used in cosmetics for over
Chemical formula C8H8O3 C9H10O3 C10H12O3 C11H14O3
Molecular weight 152.16 166.18 180.21 194.23 half a century (Weber, 1993). Rastogi et al. (1995) inves-
Melting point (C) 131 116–118 96–98 68–69 tigated the contents of parabens in cosmetic products
Boiling point (C) 270–280 297–298 – – and found a preferential use of methyl-, > ethyl-, >
Refractive index 1.525 1.505 1.505 propyl-, > butyl- > and benzyl-paraben in various cos-
pKa 8.17 8.22 8.35 8.37
metic products.
988 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
1.2.3. Pharmaceutical uses
Parabens were first used in drugs in the mid-1900s and
since they have been employed frequently in a wide
variety of formulations as preservatives. Among the para-
bens, propyl paraben is one of the most effective fungi-
stats used in pharmaceutical preparations. A variety of
drug formulations including suppositories, anesthetics,
pills, syrups, weight gaining solutions, injectable solutions
and contraceptives are known to contain parabens as pre-
servatives. Combinations of parabens are more active
than individual parabens (Boehm and Maddox, 1972).
Use concentration of parabens varies from product to
product, but seldom exceeds 1% (Neidig and Burrell,
1944; Hassler, 1954; Boehm and Maddox, 1972; Orth,
1980). Methyl paraben and propyl paraben are used as
preservatives in several over-the-counter (OTC) drugs.
The Ophthalmic Drug Panel of the US Food and
Drug Administration (FDA) has determined that
methyl and propyl paraben, if used alone and at concen-
trations effective against microorganisms, are not suit-
able as preservatives in OTC ophthalmic products,
because they are irritating to the eyes. The panel sug-
gested that further formulation studies and safety testing
be conducted on these two parabens (FDA, 1980b).
Methyl and propyl paraben have been classified as inac-
tive ingredients in dentifrices, contraceptives and topical
analgesics (FDA, 1979, 1980a,c) and in drugs for injec-
tion (via subcutaneous, intra-muscular, intra-venous,
intra-articular, intra-bursal, intra-lesional and intra-
synovial routes), inhalation and intra-nasal solutions,
ophthalmic (ointments, solutions and suspensions) and
for other routes of administration, including oral, topi-
cal, rectal and vaginal (FDA, 1996).
1.2.4. Regulatory history
Parabens are most active against yeasts and molds,
and have application in foods such as baked goods, bev-
erages, fruit products, salad dressings, syrups and olives.
Heptyl paraben is permitted by the US FDA for direct
addition to fermented malt beverages in amounts not
to exceed 12 ppm and in non-carbonated soft drinks
and fruit in various foods. Parabens are active against
Gram positive and a few Gram-negative organisms.
FDA has affirmed methyl and propyl paraben as Gener-
ally Recognized as Safe (GRAS) for direct addition to
food (21 CFR 184.1490) at concentrations up to 0.1%
and by prior sanction for indirect addition via packag-
ing materials (21 CFR 181.23). FDA has also approved
methyl-, propyl- and butyl-paraben as synthetic flavor-
ing substances and adjuvants (21 CFR 172.515) for
addition to foods at minimum quantity to beverages,
in amounts not to exceed 20 ppm. Methyl-, propyl-
and butyl paraben are permitted as direct food additives
for use as preservatives in synthetic flavoring substances
and adjuvants. As indirect food additives, methyl and
propyl paraben are permitted by prior sanction as
antimycotics in food packaging materials with no limit
or restriction. The Joint FAO/WHO Expert Committee
on Food Additives (JECFA) and the Flavor and Flavor
and Extract ManufacturerÕs Association (FEMA) have
also approved the use of methyl and propyl paraben in
food. In 1974, JECFA recommended the group Accept-
able Daily Intake (ADI) for the methyl-, ethyl-, and pro-
pyl-esters of p-hydroxybenzoic acid as 0–10 mg/kg body
weight/day (JECFA, 1974).
Use of parabens is also permitted in Japan. Of the
72,609 samples tested, Ishiwata et al. (1997) reported
detection of parabens in 903 out of 9876 food products
(9.1%). The detection rate and average concentration in
all foods in which these are permitted for use were 28.6%
and 8.8% of the limits, respectively. The highest detec-
tion rate among the foods permitted for use was soy
sauce (49.8%) and the average concentration in all tested
soy sauce samples was also the highest, 0.0342 g/kg as
p-hydroxybenzoic acid. This concentration corre-
sponded to 13.7% of the permissible limit. p-Hydroxy-
benzoic acid was detected in 16 out of 6775 samples
(0.24%) of foods in which the esters are not permitted
and the average concentration was 0.093 mg/kg as
p-hydroxybenzoic acid in the foods.
The use of parabens is permitted under the European
Union Directive No. 95/2/EC on food additives other
than colors and sweeteners. Use of parabens (methyl-,
ethyl-, propyl-, butyl-, and benzyl-; also includes iso-
butyl- and isopropyl-paraben) in cosmetic products as
a preservative with a maximum concentration of 0.8%
(w/w), calculated as p-hydroxybenzoic acid, is permitted
by the Danish and EEC regulations (Howlett, 1992;
Rastogi et al., 1995). The maximum concentration limit
set for any single ester was 0.4%. Parabens may be pres-
ent in some cosmetics labeled as ‘‘hypoallergenic’’. Sev-
eral other countries, including as Canada, Norway,
Philippines, Sweden and Switzerland, have also ap-
proved the use of parabens as antimicrobial food addi-
tives. There is a close parallel between uses of paraben
and those of sodium benzoate in the countries where
the parabens are permitted.
Per request from the European Commission, an ex-
pert science panel at the European Food Safety Author-
ity (EFSA, 2004) established the group ADI of 0–10 mg/
kg/day for methyl and ethyl parabens and their sodium
salts, but not propyl paraben, due to recent research that
questioned the effect of these parabens on sperm pro-
duction in rats. While the presence of propyl paraben
in the diet is limited and unlikely to represent a risk to
consumers, the panel was unable to recommend a spe-
cific ADI for propyl paraben based on current evidence.
The latest opinion on parabens from the EFSA panel
follows on from a 1994 evaluation by the former EC
Scientific Committee for Food (SCF) that allocated a
temporary group ADI of 0–10 mg/kg/day for methyl-,
ethyl- and propyl-paraben and their salts.
 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
1.3. Consumption
1.3.1. Exposure to parabens via food
Several methods can be applied to estimate the con-
sumption of parabens. First and foremost, the per capita
estimate of intake, also called Maximum Survey-derived
Daily Intake, is based on ‘‘disappearance data’’ of the
amounts added to food as an ingredient. In contrast
to these methods, which are based on the actual
intake of the food ingredient, the Theoretical Added
Maximal Daily Intake (TAMDI) values, such as FEMA
Possible Average Daily Intake (PADI) and Possible
Maximum Daily Intake (PMDI), are calculated on the
basis of theoretical consumption of all food to which
the ingredient has been added. In addition to its con-
sumption from food as an added ingredient, exposure
to parabens also occurs from drug intake and cosmetic
use.
Based on the FDA assumptions and the annual poun-
dage ‘‘disappearance’’ reported by the producers to NAS
(National Academy of Sciences) of 9190 lb for propyl
paraben and 933 lb for methyl paraben for the year
1987, the calculated per capita individual consumption
of propyl paraben and methyl paraben is 0.7788 mg/
day (0.01298 mg/kg/day) and 0.6 mg/day (0.01 mg/kg/
day), respectively. Lucas et al. (1999) reported a disap-
pearance value of 0.1 lb for propyl paraben and 330 lb
for methyl paraben. The ‘‘disappearance’’ value reported
for propyl paraben seems very unlikely and hence this
value is not considered for consumption estimates. Prob-
ably because the Lucas et al. (1999) data represents flavor
use only, not the bulk of the use, which is in non-flavor
products. Based on this report, the per capita consump-
tion of methyl paraben is estimated as 0.03 mg/day
(0.0005 mg/kg/day).
To determine the specific foods in which methyl and
propyl paraben were used and at what levels, a represen-
tative cross-section of food manufacturers was surveyed
(FDA, 1973). Available surveys of consumer consump-
tion were obtained and combined with the production
information to obtain an estimate of the consumer expo-
sure to methyl and propyl paraben. The survey reported
Table 2
Possible average daily intake of methyl and propyl paraben (LSRO/FASEB (1972)
Age group Possible daily intake in mg
Total
Average Maximum
Methyl Propyl Methyl
0–5 months 5 5 9 10
6–11 months 49 56 108
12–23 months 93 105 155
2–65+ years 222 238 347
a
Calculations based on an average body weight of 60 kg for an adult. For infants the estimated body weights by age group were 0–5 months, 5 kg;
6–11 months, 8 kg; 12–23 months, 11 kg.
989
the total methyl paraben used in food in 1970 to be
about 16 times that used in 1960.
A National Research Council subcommittee supplied
the information on the possible daily human intake of
methyl and propyl paraben to LSRO/FASEB (1972).
The possible intake per kg of body weight for different
age groups is presented in Table 2. The LSRO/FASEB
committee recognized the calculated daily intake of
parabens per kg body weight in the age groups 2 to
65+ years could be deceptively low, because individuals
from age 2 to maturity obviously weigh less than 60 kg;
thus the daily intake of parabens per kg for children
could be significantly higher than indicated.
The FEMA PADI (Possible Average Daily Intake)
employs the usual use level values and mean consump-
tion values (based on Market Research Corporation of
America mean frequency of eating and USDA mean
portion size of 34 general food categories) (MRCA,
1965) to determine the consumption. The FEMA PADI
for methyl and propyl paraben was determined as
0.22 mg/day (0.004 mg/kg/day) and 0.237 mg/day
(0.004 mg/kg/day), respectively. A possible maximum
daily intake (PMDI) can be calculated using the mean
consumption of food as above and the maximum levels
proposed by FEMA. The PMDI calculated theoretical
value for propyl paraben was 0.2518 mg/day or
0.0042 mg/kg/day for an average individual weighing
60 kg.
The major uses of the parabens in the food industry
are cakes, pie-crusts, pastries, icings, toppings and fill-
ings (0.03–0.06% of a combination of 3:1 methyl and
propyl parabens); soft drinks (0.03–0.05% of a 2:1 ratio
of methyl and propyl parabens); creams and pastes
(0.1% of a combination of parabens); jams, jellies and
preserves (0.07% of a 2:1 ratio of methyl and propyl
parabens); olives and pickles (0.1% of a combination
of parabens); syrups (0.07%). Available recent surveys
of consumer consumption were combined with the max-
imum permissible levels to obtain a current estimate of
the consumer exposure to parabens (Table 3). As per
the USDA food consumption data for year 1989–1991
(Krebs-Smith et al., 1997) the age group 20–39 are the
Per kg body weighta
Average Maximum
Propyl Methyl Propyl Methyl Propyl
1 1 2 2
124 6 7 14 16
179 8 10 14 16
381 4 4 6 6
990 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
Table 3
Parabens consumption via food
Food Mean daily Level
consumed of
over three use (%)
day period
(90th percentile)
(g)a
Cake 161 0.06
Piecrust 274 0.06
Doughnuts and sweet rolls 138 0.06
Syrup 130 0.07
Pickles 64 0.10
Jelly, jams, marmalade 559 0.07
Total paraben consumption
a
USDA, ARS, NFS Report No. 91-3 of food consumption for
1989–1991.
highest consumers of jelly, jams, preserves and marma-
lade with an intake of 56 g/day in males and 36 g/day
in females, at the 90th percentile. The amount of methyl
paraben consumed at the 90th percentile in jams and jel-
lies at a level of 0.07% is 39 and 25 mg/day for males and
females, respectively. While the amount of propyl para-
ben consumed at 90th percentile in jams, jellies, pre-
serves and marmalade at a level of 0.1% is 56 and
36 mg/day for males and females, respectively.
Data presented in Table 3 shows mean daily con-
sumption of food products would result in a paraben in-
take of 466 mg/day, a gross exaggeration considering the
amount of parabens manufactured each year
(<17,000 lbs). The unlikelihood of such a grossly exag-
gerated consumption occurring is confirmed by the pro-
pyl paraben disappearance data, which indicates the
total amount of propyl paraben used in 1987 was
15,316 lb, with per capita consumption of 0.779 mg/
day. Methyl-, butyl- and heptyl-parabens have even
lower rates of consumption, estimated by FDA/CFSAN
to be 0.063 mg/day, 37 ng/day and 0.032 mg/day, respec-
tively. No data are available for ethyl paraben. There-
fore, based on disappearance data, consumption of all
parabens is unlikely to exceed 1 mg/day, a far smaller
amount than might be calculated from maximum levels
in all permitted food categories. In a similar attempt to
calculate the paraben consumption, the Select Commit-
tee on GRAS Substances (SCOGS) in 1972 also con-
cluded that consumption was exaggerated. The
SCOGS estimated the daily average intake of both
methyl and propyl paraben combined, would be
0.15 mg (SCOGS, 1972). Paraben intake from natural
sources is negligible.
1.3.2. Exposure to paraben via cosmetics and drugs
Parabens are widely used in cosmetics and are well
absorbed through the skin; hence a calculation of the
total body burden of parabens should include cosmetics
and personal care products and drugs. Elder (1984) re-
ported that approximately 10% of these types of prod-
Mean daily ucts and drugs contain parabens at levels not greater
amount than 1%. If the daily maximum amount of cosmetic
paraben
consumed
and personal product use is estimated to be 50 g and
over 10% of these contain parabens at the highest level, pos-
three-day sible exposure is 50 mg/day or 0.833 mg/kg/day.
period (mg) In addition to cosmetics and food products, drugs
96 also contain parabens as an antimicrobial preservative.
164 An estimate of exposure should also include exposure
82 via drugs, including those given parenterally and by
91
6
the oral, topical and vaginal routes. Maximum use levels
27 in drugs are 2–10-fold lower than levels used in cosmet-
ics. Therefore, assuming a maximum level of 0.5% and
466
the volume of all drugs taken or applied in a single
day is approximately 20 g (maximum), possible expo-
sure of parabens via drugs is 0.1 g. However, because
greater than 50% of drugs are not taken on a daily basis
and if parabens are used in 50% of the drugs marketed,
paraben consumption via drugs is reduced to 25 mg/day
or 0.417 mg/kg/day.
1.3.3. Consumption summary
Data are available for methyl and propyl paraben per
capita consumption, as well as possible average daily in-
take. Based on the FDA reported disappearance data,
the average person is estimated to consume as much as
0.6 mg/day (0.01 mg/kg/day) and 0.78 mg/day (0.013
mg/kg/day) of methyl and propyl paraben, respectively
for an individual weighing 60 kg. The FDA estimate
of consumption for methyl and propyl paraben based
on disappearance data is higher than the FEMA PADI
value for these parabens of 0.22 mg/day (0.004 mg/kg/
day) and 0.237 mg/day (0.004 mg/kg/day), respectively.
In addition to food products, parabens are also con-
sumed via cosmetics and personal care products
(0.833 mg/kg/day) and drugs (0.417 mg/kg/day). The
total consumption of parabens from all sources is esti-
mated as 75.78 mg/day or 1.26 mg/kg/day for an individ-
ual weighing 60 kg. On the basis of the above estimates,
total daily paraben exposure is 76 mg: with food
accounting for approximately 1 mg/day; cosmetics and
personal care products, 50 mg/day; and drugs, 25 mg/
day.
2. Biological data
2.1. Absorption, metabolism and excretion
In an evaluation of the health aspects of methyl and
propyl paraben, the Select Committee on GRAS Sub-
stances (SCOGS) reported that in rats, rabbits, dogs,
cats and humans, parabens (methyl and propyl) are ab-
sorbed from the gastrointestinal tract and metabolized
(SCOGS, 1972). Neither methyl nor propyl parabens
 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
are accumulated in the body. The major metabolites ex-
creted in urine, in decreasing order, were reported as
p-hydroxybenzoic acid and the glycine, glucuronic acid
and sulfuric acid conjugates of p-hydroxybenzoic acid.
The SCOGS report also stated that most, but probably
not all of the ingested parabens, were metabolized to the
foregoing substances through normal pathways in the
liver and kidney.
In a series of studies, Tasukamoto and Terade (1960,
1962, 1964) investigated the metabolic fate of methyl-,
ethyl-, propyl- and butyl-paraben in rabbits. A single
dose (0.4 or 0.8 g/kg) of methyl paraben was adminis-
tered to rabbits by gavage. Thirty-nine percent of the
administered methyl paraben dose was excreted as free
p-hydroxybenzoic acid, while the rest appeared as gly-
cine (15%), glucuronic acid (ester 7% and ether 15%)
and sulfuric acid (10%) conjugates of p-hydroxybenzoic
acid. Urinary excretion of metabolites was rapid, with
86% cleared in 24 h. The rate of excretion was slightly
lower with the larger dose. Oral administration of
methyl-, ethyl-, propyl- and butyl-paraben at a dose
of 0.4 and 0.8 g/kg resulted in excretion of 0.2–0.9% of
the unchanged ester by 24 h. As the length of the alkyl
chain increases, the rate of urinary excretion of
p-hydroxybenzoic acid decreases. In general, 24 h after
paraben administration, 25–39% was excreted as
p-hydroxybenzoic acid, 25–29% as the glycine conjugate,
5–8% as the ester glucuronide, 10–18% as ether glucuro-
nide and 7–12% as the sulfate.
In another study, Jones et al. (1956) investigated the
pharmacokinetics of methyl-, ethyl-, propyl-, or butyl-
paraben (individually) in dogs by the intra-venous
(50 mg/kg) and oral (1 g/kg) routes. The concentrations
of parabens and their metabolites in blood and urine
were determined at different time points. Immediately
following intra-venous administration of parabens, very
little parent compound remained in the blood. The
p-hydroxybenzoic acid metabolite was detectable in the
blood at 6 h following intra-venous injection and at
24 h following oral ingestion. Recovery of all parabens,
except butyl paraben, ranged from 58% to 94% of the
administered dose. The recovery of butyl paraben was
40% and 48% after oral and intra-venous administration,
respectively. In dogs given 100 mg/kg/day methyl-,
ethyl-, propyl-, and butyl-paraben by the intra-venous
route, pure ester was recovered in the brain, spleen and
pancreas. High levels of metabolites were detected in
the liver and kidneys. Dogs given 1 g/kg/day of methyl
or propyl paraben for one year by the oral route did
not show any evidence of paraben accumulation. Over
the course of the study, the rate of urinary excretion of
paraben and their metabolites increased until 96% of
the dose was excreted per day.
Derache and Gourdon (1963) studied the pharmacol-
ogy of methyl-, ethyl- and propyl-paraben in rats after
oral administration of 100 mg of ester. Administration
991
of parabens in rats resulted in quick absorption from
the gastrointestinal tract. The absorbed parabens were
readily hydrolyzed to p-hydroxybenzoic acid by ester-
ases in different organs. Blood and urine analysis of
the parent compound, as well as its metabolites, was
performed at different time points. As early as 30 min
after dosing, paraben metabolites, but not the parent
compound, were detected in the urine. One and a half
hour following dosing, excretion of metabolites was
maximal and decreased thereafter. p-Hydroxyhippuric
acid was detected in the urine within 30 min of adminis-
tration and its concentration in the urine increased dur-
ing the next 4 h. The glucuronide and ethereal sulfate
metabolites appeared between 30 and 75 min post-inges-
tion. Within 90 min, 67–75% of the total paraben dose
was excreted as p-hydroxybenzoic acid and 8–9% as glu-
curonyl derivatives. During the first hour of administra-
tion a continuous rise in p-hydroxybenzoic acid
occurred, but thereafter, the concentration decreased
and leveled off 1–2 h after ingestion. The concentration
in the blood remained extremely low. The investigators
suggested that there were two stages of paraben detoxi-
fication, the first being rapid absorption of paraben and
excretion of p-hydroxybenzoic acid in urine and the sec-
ond, metabolic detoxification by glucuronic-, sulfo-, and
glycino-conjugation.
Phillips et al. (1978) studied the metabolic fate of 14C
ring labeled ethyl and propyl paraben administered to
male cats at a dose level of 156 and 158 mg/kg orally.
Urine was collected at 24, 48 and 72 h following the
administration and feces were collected at 72 h. By the
end of 72 h, 96% of the label was recovered. Approxi-
mately 90% label was recovered in the 24 h urine, while
6% and 3% were recovered in feces by the end of 24 h,
respectively. Urine analysis revealed the presence of
p-hydroxybenzoic acid and p-hyroxyhippuric acid as
the two major metabolites. The investigators concluded
that within 72 h of oral administration, both parabens
were completely excreted and the major urinary meta-
bolites were p-hydroxyhippuric acid and p-hydroxyben-
zoic acid.
In vitro studies suggest that esterases in the liver and
kidneys were extremely efficient in hydrolyzing parabens
(100% hydrolysis in 3 min). In an in vitro study using rat
skin with and without the esterase inhibitor isofluro-
phate, Bando et al. (1997) studied the role of skin
metabolism on percutaneous penetration of propyl
paraben. In the absence of the esterase inhibitor isofl-
urophate, application of propyl paraben resulted in
approximately 30% absorption in intact form. Isoflurate
did not significantly decrease the amount that pene-
trated after the application of propyl paraben. In a per-
cutaneous absorption study, Whitworth and Jun (1973)
investigated the absorption of parabens in frogs. Frogs
were immersed in the solution of parabens. Uptake of
the parabens increased as the length of ester carbon
992 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
chain increased. During the first 20 min of immersion,
absorption was fastest. The results of this study indicate
that the greater the lipid solubility of the paraben, the
higher the rate of absorption.
In another in vitro study, Dal-Pozzo and Pastori
(1996) studied percutaneous absorption through human
skin of a series of six parabens (methyl-, ethyl-, propyl-,
butyl-, hexyl- and octyl-paraben) from cosmetic formu-
lations. A diffusion chamber was used, where the receiv-
ing phase was a physiological solution of albumin.
Maximum fluxes and permeability constants of the
parabens were determined from different vehicles repre-
senting hydrophilic and lipophilic phases, and from dif-
ferent types of common commercial cosmetic emulsions,
containing a known quantity of a paraben. Depending
on the partition coefficient of the permeant, composition
and time of storage of the emulsion, significant skin
absorption was noted. Except for methyl paraben,
where fluxes from all types of the emulsions were higher,
fluxes decreased with increasing lipophilic character of
the paraben.
In several studies, absorption of butyl paraben from
ointments was reported from mouse skin. In a percuta-
neous absorption study of butyl paraben (0.015–0.1%
aqueous) through guinea pig skin, Komatsu and Suzuki
(1979) reported that in the presence of solublizers, such
as polysorbate 80, polyethylene glycol or PEG 400,
absorption of butyl paraben was reduced but the antimi-
crobial activity was increased. The amount of butyl
paraben that passed through the skin was dependent
on the partition coefficient and the presence of
solublizers.
In an in vitro study, Cross and Roberts (2000) deter-
mined the effect of occlusion on epidermal penetration
of parabens from a commercial allergy test ointment,
acetone and ethanol vehicles. Parabens were applied at
a dose of 5 mg/cm2 to epidermal membranes mounted
in Franz-type diffusion cells. At 2, 4, 6, 8 and 10 h, the
receptor phase (20% ethanol) was completely removed
and replaced. In order to effect occlusion, immediately
after dosing, a 20 lm piece of high-density polyethylene
was placed over the application site. The amount of
paraben in receptor fluid and in the epidermis was deter-
mined. A significant change in the epidermal flux of
parabens from each of the vehicles following occlusion
was noted. A decrease was noted following occlusion
of the ointment formulation, while increases were ob-
served for the acetone and ethanol vehicles. These stud-
ies suggest that the effects of occlusion are strongly
vehicle dependent.
In a two layer skin diffusion/metabolism model, Seko
et al. (1999) investigated the effect of cutaneous metab-
olism on the skin penetration of drugs. Permeation of
propyl- and butyl paraben was conducted with and
without the esterase inhibitor, diisopropyl fluorophos-
phate. Pre-treatment of skin with the esterase inhibitor
prolonged the lag time for the penetration. The inhibitor
significantly decreased the total flux of butyl paraben,
but not that of propyl paraben. In another study, Wooi
et al. (1998) reported in vitro application of propyl para-
ben to rat skin resulted in 70% of the total penetrant
amount appearing as a metabolite. Treatment of skin
with the esterase inhibitor, diisopropyl fluorophos-
phates, completely inhibited the appearance of the
metabolite, but did not inhibit the penetration of propyl
paraben.
Exposure of freshly isolated rat hepatocytes to propyl
paraben and diazinon, an inhibitor of the (esterase) en-
zyme responsible for the breakdown of propyl paraben,
enhanced and accelerated cell killing as well as the loss
of cellular ATP and total adenine nucleotides (Nakag-
awa and Moldeus, 1998). In a comparative toxic effect
study, butyl and isobutyl parabens were more toxic than
propyl-, isopropyl parabens, while ethyl and methyl
parabens and p-hydroxybenzoic acid were less toxic
than propyl paraben. The metabolites, p-hydroxyben-
zoic acid or the alcohol derived from propyl paraben,
did not exhibit any significant toxicity. The increased
toxic action of propyl paraben in the presence of ester-
ase inhibitor suggests that propyl paraben itself, rather
than its hydrolysis product, was responsible for the tox-
icity observed. These studies also suggest that parabens
are hydrolyzed enzymatically by hepatic carboxyester-
ases, a group of b-esterases that act upon a wide range
of xenobiotic ester substrates.
In a human volunteer given 2 g of propyl paraben
daily for 5 days, 17.4% of the administered dose was ex-
creted in the form of p-hydroxybenzoic acid, of which
3.7% paired with glycine and 13.7% was glycine-
free (Sabalitschka and Neufeld-Crzelliter, 1954). The
authors reported that 55% of the excreted propyl para-
ben was conjugated with sulfuric acid. The parent com-
pound was not found in the urine. In this experiment,
the authors were unable to account for all of the in-
gested propyl paraben and therefore concluded that
breaking of the benzene ring must occur to some extent.
In another study, Heim (1960) administered six human
subjects 10 or 20 mg/kg body weight of the propyl para-
ben orally. At 60, 135 and 255 min, p-hydroxybenzoate,
but not the ester, was detectable in serum. The maxi-
mum serum level of p-hydroxybenzoate attained in ser-
um was 4.5 lg/ml.
Recently, Darbre et al. (2004) measured concentra-
tions of esters of paraben in human breast tumor and
suggested that at least a proportion of the parabens
present in cosmetic, food and pharmaceutical products
can be absorbed and retained in the body tissue without
hydrolysis. In this study mean concentrations for
methyl-, propyl-, butyl-, ethyl- and isobutyl-paraben
were reported as 12.7, 2.6, 2.3, 2.0 and 0.9 ng/g tissue,
respectively. These studies are further discussed below
(see Section 2.3.6.2).
 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
2.1.1. Biochemical/pharmacological effects
Parabens are reported to bind to serum bovine albu-
min. Binding increases with increased alkyl ester chain
length of the paraben. The binding process is endother-
mic and hydrophilic in nature. Binding of paraben with
protein results in loss of its antifungal activity. Using
a fluorescent probe, it has been documented that the
side chain of the paraben is the primary binding site to
bovine serum albumin (Jun et al., 1971). At a concentra-
tion of 400 lg/ml, methyl and propyl paraben competi-
tively inhibited bilirubin binding to serum albumin. In
another study, Rasmussen et al. (1976) reported that,
while methyl and propyl paraben bind to serum albu-
min, only methyl paraben displaces bilirubin from albu-
min. The serum albumin binding constant increases
significantly from propyl paraben to butyl paraben
(Otagiri and Perrin, 1977).
In an in vitro study, methyl-, ethyl- and propyl-para-
ben were found to competitively inhibit the oxidation of
ethanol and reduction of acetaldehyde. These parabens
were found to competitively inhibit all three human
aldehyde dehydrogenase isozyme classes; propyl para-
ben was the most effective (Dingley et al., 1985). Inhibi-
tion of aldehyde dehydrogenase substantially affects
ethanol metabolism and suitable inhibitors could be
valuable for the study of ethanol metabolism and pre-
ventive therapy of methanol and ethylene glycol poison-
ing. However, it is not clear whether parabens inhibit
ethanol oxidation in vivo.
In another in vitro study, both methyl and propyl
paraben induced dose-dependent, rapid, reversible
relaxation of the isolated guinea pig smooth trachea
(Geddes and Lefcoe, 1973). At a dose of 1.5 lg/ml, pro-
pyl paraben potentiated the effects of isoproterenol and
dibutyryl cyclic AMP. The investigators of this study
suggested that the bronchodilation effect of parabens
might be due to the inhibition of phosphodiesterase.
2.2. Toxicological studies
2.2.1. Acute toxicity studies
Low acute oral toxicity has been demonstrated for
parabens in laboratory animals. Acute toxicity of para-
bens decreases as the alkyl chain length increases. Mat-
Table 4
LD50 (mg/kg) of parabens in different animal species by different routes
Species Route Methyl paraben
Mouse Oral >8000
Mouse Oral (sodium salt) 2000
Mouse Subcutaneous 125–1200
Mouse Intra-peritoneal 760–960
Rat Oral 2100–8000
Rabbit Oral 3000a
Dog Oral 3000a
a
LD100 or lethal dose.
993
thews et al. (1956) suggested that the decrease in toxicity
with increasing chain length was due to longer hydroly-
sis time. Acute toxicological effects of parabens from dif-
ferent published reports are summarized in Table 4.
2.2.1.1. Oral acute toxicity. Matthews et al. (1956)
determined the oral LD50 doses of several parabens
and their sodium salts in mice. The fatal doses of para-
bens and its sodium salt in mice produced rapid loss of
muscular control (ataxia), deep depression of the central
nervous system and rapid death. Recovery from a non-
fatal dose occurred quickly (Matthews et al., 1956).
Schuebel (1930) studied the acute toxicity of orally
administered doses of parabens in dogs and rabbits.
The author of this study reported that toxicity of para-
bens decreased as the alkyl chain length increased. In an
acute toxicity study in dd-strain mice, Sado (1973) re-
ported 6.322 g/kg as the LD50 dose of propyl paraben.
Studies on the toxicity of mixtures of paraben did not
exceed theoretical (additive) values, thus suggesting that
these compounds do not exhibit synergistic toxicity. In a
study conducted to determine the minimum lethal dose
(the smallest dose required to induce 60–80% mortality),
a 60:40 mixture of sodium salts of propyl- and ethyl
paraben was administered orally to groups of 5–10 gui-
nea pigs at doses of 4.75–6.0 g/kg. Animals were ob-
served for 10 days after the treatment. Doses of 5.0
and 6.0 g/kg induced 60% mortality, although surviving
animals became progressively worse with increasing
doses. The minimum lethal dose was determined as
5.0 g/kg (Applied Research Laboratories, 1939).
Product formulations containing various concentra-
tions of methyl-, ethyl-, propyl- or butyl-paraben were
tested for acute oral toxicity in rats (Elder, 1984). In sev-
eral of these studies, products containing high levels of
parabens (in the range of 5–25 g/kg), when administered
by gastric intubation to rats did not cause deaths.
2.2.1.2. Subcutaneous and intra-peritoneal acute toxi-
city. In an early study, cited in Elder (1984), subcutane-
ous administration of methyl paraben at doses greater
than 165 mg/kg, resulted in a transient exhaustion, ataxia
and respiratory distress. Because of solubility limita-
tions, the effects of methyl paraben were studied at
Ethyl paraben Propyl paraben Butyl paraben
6322 to >8000 13,200
1500 3700 950
1650
640
4300 >8000
4000a 6000a
5000a 4000a
994 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
doses up to 333 mg/kg. In a study on the effects of so-
dium salts of methyl-, ethyl-, propyl-, and butyl-paraben
in mice, the acute LD50 by subcutaneous route was re-
ported as 1.20, 1.65, 1.65 and 2.5 g/kg, respectively (Al-
der-Hradecky and Kelentey, 1960). Matthews et al.
(1956) determined the intra-peritoneal LD50 of several
parabens and their salts in mice. The authors of this
study also noted that intra-peritoneal injection of
400 mg/kg of propyl paraben in the mouse produced a
reversible paralysis. These studies are summarized in
Table 4.
2.2.1.3. Intra-venous acute toxicity. In a series of studies,
Matthews et al. (1956) investigated the effects of intra-ve-
nously administered methyl and propyl paraben in mice
and dogs. The acute LD50 of the sodium salt of methyl
and propyl paraben in mice by the intra-venous route
was 170 and 180 mg/kg, respectively. At doses of approx-
imately 80 mg/kg, methyl paraben caused paralysis in
mice and precipitous but brief falls in blood pressure in
dogs. Propyl paraben caused paralysis at doses of
approximately 50 mg/kg in mice, and precipitous, but
brief, falls in blood pressure in dogs given approximately
65 mg/kg. The authors also studied the effects of intra-ve-
nously administered doses (1–1400 mg/kg) of methyl and
propyl paraben on the cardiovascular and autonomic
nervous systems. A sharp, but brief fall in the blood pres-
sure and a corresponding rise in the jugular venous pres-
sure after the administration of paraben were noted.
Mortality was associated with the hypotensive action.
A correlation was observed between the rate of injection
and cardiovascular effects. No effects of paraben were
noted on the nervous system.
In vitro studies with paraben preservatives have been
shown to cause vasodilatation in human pial cortical
arteries and endothelium of dog artery (Brandt et al.,
1983; Hamilton et al., 1990). However, in vivo model
studies in cats by Pompy et al. (1991) suggest that propyl
paraben (0.1 mg/kg) and methyl paraben (0.9 mg/kg)
given through the cephalic vein did not cause or sub-
stantially augment the intra-cranial pressure increases
associated with administration of paraben-containing-
injections of succinylcholine.
2.2.2. Irritation studies
2.2.2.1. Dermal irritation. Application of pastes contain-
ing hydrophilic ointment and either 10% methyl or
propyl paraben to the shaved backs of albino rabbits
for 48 h did not cause irritation. At the end of 48 h,
analysis of the kidney tissue did not reveal the presence
of the parent compound or its metabolites (Sokol,
1952). Results of several irritation studies have been re-
ported by the Cosmetic, Toiletry and Fragrance Asso-
ciation (CTFA). Methyl-, propyl- and butyl-paraben
have been tested alone or in product formulations in
the Draize skin irritation technique in rabbits. Undi-
luted methyl paraben was found to produce mild skin
irritation, while product formulations containing
methyl paraben produced none to mild irritation.
There was no relation between the concentration of
methyl paraben and degree of irritation. Ethyl paraben,
diluted or undiluted, produced no sings of irritation. In
one report, a product formulation containing 0.3%
propyl paraben was applied daily to the shaved skin
of albino rabbits for four days. Minimum irritation
with a maximum primary irritation index (PII) of 0.5
(maximum score 4) was noted (CTFA, 1977). Another
product containing 0.2% propyl paraben produced
minimal irritation with a PII of 0.5 (Leberco Laborato-
ries, 1978a). Yet another product containing both 0.2%
methyl paraben and 0.1% propyl paraben was mini-
mally irritating with a PII of 0.5 (CTFA, 1979). No
signs of irritation with a product formulation contain-
ing 0.2% propyl paraben and 0.1% butyl paraben were
noted (CTFA, 1980c). A formulation containing 0.2%
propyl paraben and 0.1% butyl paraben was applied
to the genital mucosa of six albino rabbits. The single
0.1 ml application of the undiluted product produced
no evidence of mucosal irritation during the seven
day observation period (CTFA, 1980c).
2.2.2.2. Eye irritation. Several studies in rabbits have in-
vestigated the eye irritation effects of products containing
methyl-, ethyl-, propyl- and/or butyl-paraben at concen-
trations of 0.1–0.8%. Most products produced no signs
of eye irritation (Leberco Laboratories, 1978b; CTFA,
1979). Other products produced slight or minimal eye irri-
tation, with scores of 1–3.3/110 (Stillmeadow, 1978;
CTFA, 1980c, 1981a).
2.2.3. Sensitization studies
Sokol (1952) investigated the sensitizing effects of
methyl-, ethyl-, propyl- and butyl-paraben (0.1% in sal-
ine) in guinea pigs. Animals were intra-cutaneously
administered parabens (0.1% in saline) three times
weekly for three weeks (total of 10 injections). At 24 h
after the first injection, no reaction was observed. Simi-
larly, a challenge injection two weeks after the last injec-
tion into an adjacent site did not show any allergic
response. In a similar type of experiment following the
Draize protocol, Matthews et al. (1956) did not find
any reaction from an intra-cutaneous injection of
methyl-, ethyl-, propyl- and butyl-paraben (0.1% in sal-
ine) into the shaved dorsal skin of guinea pigs. The
authors of this study concluded that parabens are non-
sensitizing. In a maximization test with a multiple dose
design in guinea pigs, Andersen et al. (1995) did not find
any significant sensitization potential with propyl
paraben.
In a murine local lymph node assay (LLNA), Basket-
ter et al. (2001) studied several chemicals that can act
as contact allergens. In this study, propyl paraben was
 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
reported as non-sensitizing. A methyl/propyl paraben
system was also reported negative in this assay (Basket-
ter et al., 1994). In another study, Basketter et al. (1999)
determined the threshold of 134 chemicals, including
propyl paraben, for classification as skin sensitizer. Pro-
pyl paraben was reported negative in the LLNA assay
and the guinea pig maximization test (GMPT). Marzulli
et al. (1968) studied the delayed contact hypersensitivity
of methyl and propyl paraben in dinitrochlorobenzene
(DNCB) hypersensitive guinea pigs. None of the
DNCB-sensitive animals were sensitized to 5% methyl
paraben or 3% propyl paraben by the intra-dermal route
at induction or by intra-dermal or topical routes at chal-
lenge. In another sensitizing potential study of butyl
paraben (Brulos et al., 1977), six of the 20 animals tested
reacted to the challenge patch. The mean erythema score
was 1.7 (maximum score 4). Microscopic examination of
tissue from two of the six animals showed lesions con-
sidered allergic.
2.2.4. Short-term and subchronic toxicity studies
The Cosmetic Ingredient Review (CIR) expert panel
reviewed several short-term and subchronic studies on
parabens submitted by the Cosmetic, Toiletry and
Fragrance Association (CTFA). The majority of the
studies submitted by CTFA were not published and
remain proprietary, but CIR briefly described these
reports in their evaluation (Elder, 1984). In an oral
study, a product formulation containing 0.2% methyl
paraben and 0.2% propyl paraben at doses of 0, 40
or 200 mg/kg/day for one month in rats (10/sex/group)
was studied. The test material was prepared as 2% or
10% dispersion in corn oil and administered daily in
dose volumes of 2 ml/kg. Controls received an equal
volume of corn oil. During the course of study, one
high dose male rat died. The dead rat had pneumonia,
presumably caused by test material accidentally placed
in the trachea. No signs of toxicity were noted in the
surviving rats. Body weight gain and food consump-
tion were unaffected by the treatment. Slight changes
noted in hematological and blood chemistry values
and organ weights were not biologically significant.
Histological examination of the tissues revealed no
treatment-related changes (CTFA, 1980b).
In another study, the effects of a formulation contain-
ing 0.2% propyl paraben and 0.1% butyl paraben were
tested in male and female rats for one month. No signs
of toxicity were noted. Food consumption, body weight
gain and hematological values were similar for both the
treated and control groups. Minor changes noted in
blood chemistry and organ weights were of no toxico-
logical significance. Histological examination of the tis-
sues revealed no treatment-related changes (CTFA,
1980a).
In an early report, Bijlsma (1928) investigated the ef-
fects of methyl paraben administration at a dose level of
995
18 mg/kg/day to a dog for 28 days and at 53 mg/kg/day
to another dog for 4 days. The animals were killed at the
end of the study. No toxicity was reported, and no gross
lesions were noted upon necropsy. In rabbits, oral
administration of 0.5 g/kg/day of methyl paraben for
6 days was without obvious toxic effects. Overt toxicity
was reported at 3 g/kg/day (Elder, 1984).
In a feeding study, ethyl paraben was administered
orally to rats (5/sex/group) at dose levels of 0.2%, 1%
and 2% in diet for 25 weeks (Sado, 1973). During the
test, no significant differences in appearance, behavior,
food consumption, mortality or survival times were
noted between the control and experimental groups. In
male rats fed 0.2% ethyl paraben, significant increases
in mean body weight during weeks 22–25 were noted.
In male rats fed 1% and 2% ethyl paraben, significant
decreases in body weights were noted. Throughout the
study, values for erythrocyte numbers, hemoglobin,
hematocrit and white blood cells were normal in all ani-
mals throughout the study. No microscopic or macro-
scopic abnormalities were noted.
Inai et al. (1985) fed groups of mice (10/sex/group—
treatment and 20/sex/group—control) a diet containing
0%, 0.6%, 1.25%, 2.5%, 5% and 10% isobutyl paraben
for six weeks. All mice in the two high dose groups died
during the first two weeks of the study. A decrease in
body weight of about 10% was noted in mice fed 1.25
and 2.5% of isobutyl paraben in diet. In animals fed
the lowest dose of isobutyl paraben, the increase in body
weight gain was similar to the control group. Histolog-
ical examination revealed atrophy of the spleen, thymus
and lymph nodes, in groups dosed with P1.25% iso-
butyl paraben. Multifocal degeneration and necrosis of
the hepatic parenchyma was also noted in these groups.
No significant lesions were found in mice dosed with
0.6% isobutyl paraben.
In a dermal toxicity study, the effects of daily dermal
exposure for 3 months of a product formulation con-
taining 0.2% methyl paraben and 0.2% propyl paraben
were investigated. Rabbits (6/sex/group) were divided
into two untreated control and three treatment groups.
The formulation was administered at doses of 2 and
6 mg/cm2 over 10% of body surface area. Rabbits in
one control group and 6 mg/cm2 treated group were ex-
posed daily to one-half the minimal erythema dose of
ultraviolet light (4 min at 6 in. from Westinghouse FS-
20 lamps producing a continuous spectrum from 2800
to 4000 Å). The formulation caused persistent moderate
erythema, slight edema and mild desquamation. Occa-
sional epidermal fissures with bleeding and papuloeryt-
hema were observed. The high dose was slightly more
irritating than the low dose. Ultraviolet light did not af-
fect the severity of the irritation. During the course of
the study, two test animals died, but the deaths were
unrelated to the treatment. Food consumption, body
weight gain, hematology, blood chemistry and urine
996 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
analysis values did not reveal any significant alterations
of toxicological significance. Histological examination
of skin revealed mild to severe dermal inflammation
and hyperkeratosis with acanthosis (CTFA, 1981b).
In another dermal toxicity study in rats, the effects of
daily exposure to a product formulation containing
0.7% methyl paraben and another containing 0.3% pro-
pyl paraben were studied for 13 weeks. Animals were
treated topically with daily doses of 4.12 g/kg. The for-
mulation was applied to the anterior shaved skin, which
represented 10–15% of the total body surface area. In
the treatment group, body weight gain was significantly
decreased. Slight changes of no toxicological signifi-
cance in hematological and blood chemistry parameters
and organ weights were noted. Significant gross and
histopathologic changes were limited to the treated skin
site. The report concluded that there were no cumulative
systemic toxic effects from the formulation (CTFA,
1981c).
In yet another dermal toxicity study, a product for-
mulation containing 0.2% methyl paraben was applied
topically to rabbits for 3 months. Albino rabbits (5/
sex/group) received daily doses of 5.5 mg/cm2 applied
to 8.4% body surface area. An untreated group (7/sex)
served as a control. The product caused persistent well
defined to moderate erythema, slight edema and inter-
mittent slight desquamation. Three test animals died
during the study of conditions unrelated to treatment.
Body weight gain, food consumption, hematological
and blood chemistry values were unaffected by treat-
ment. The presence of glucose and blood in urine of
some untreated and treated rabbits was considered not
relevant. Histopathological examination of tissues of
all animals was negative for treatment-related changes
other than mild inflammation at the application site
(Elder, 1984). In addition to this study, Elder (1984)
has also described a dermal subchronic studies on
methyl paraben at dose level of 6.6 and 11 mg/cm2/
8.4% body surface area. No treatment related changes
other than mild inflammation at the application site
were found.
Neostigmine formulations contain methyl and propyl
paraben as preservatives. Several investigators studied
the safety of the intra-thecal administration of neostig-
mine containing methyl and propyl paraben (Gurun
et al., 1997). The studies in rats and sheep did not show
any evidence of neurotoxicity from intra-thecal injection
of neostigmine containing methyl and propyl paraben.
Similarly, administration of neostigmine containing
glucose, methyl paraben and propyl paraben did not
produce behavioral, chemical or histopathological evi-
dence of neurotoxicity.
2.2.5. Chronic toxicity/carcinogenicity studies
2.2.5.1. Chronic toxicity studies. In a 96-week study,
Matthews et al. (1956) investigated the effects of methyl
and propyl paraben in rats. Rats (24/sex/group) were fed
diets containing 2% or 8% for 96 weeks. Additionally,
ethyl and butyl paraben were fed to the same numbers
of rats at concentrations of 2% and 8% in the diet for
12 weeks. Negative controls were included in the study.
Food intake and the body weights of animals were
recorded every other week. Based on the food intake,
biweekly paraben consumption was determined. Food
and paraben intake remained fairly constant throughout
the course of experiment. Intake of methyl and propyl
paraben for rats on the 2% diets averaged between 0.9
and 1.2 g/kg body weight/day and for the 8% diet, aver-
aged from 5.5 to 5.9 g/kg body weight/day. At the end of
the experiments, all animals were killed and kidney,
liver, heart, lung, spleen and pancreas were removed
for microscopic examinations. Rats maintained on diets
containing 8% methyl or propyl paraben showed only
decreased weight gain. The only effect noted was a de-
crease in the body weight during the early part of the
study and this decrease did not appear to be related to
the treatment because the initial decrease in weight dis-
appeared with time while the consumption of methyl
paraben as well as food intake remained constant
throughout the course of the experiment. Rats fed a diet
containing 2% methyl or propyl paraben did not show
any toxic effects. Necropsies at the conclusion of the
experiments did not show any treatment related abnor-
malities. These studies indicate a NOAEL of 5.5 g/kg/
day.
In another study, Matthews et al. (1956) fed 0.5 or
1.0 g/kg/day methyl or propyl paraben by capsule,
6 days/week to weanling dogs (3/group) for about a
year. Two untreated dogs served as controls. On com-
pletion of the study, all dogs were killed for necropsy.
Parabens did not show any toxic effects. All animals
were in excellent condition throughout the experiment.
In this study, only six organs were examined microscopi-
cally and all tissues were normal. In another brief cita-
tion, two dogs were unaffected by oral methyl or
propyl paraben levels of 0.5 g/kg/day, but evidence of
toxicity appeared at 4 g/kg/day of propyl paraben
(FDA, 1973).
Elder (1984) described a study in which a 60:40 mix-
ture of sodium salts of propyl- and ethyl paraben,
respectively, was fed to 40 rats for 18 months. The ani-
mals received 0.014 g/kg/day of the mixture. At 2 and
4 months of feeding, 10 rats each were killed for nec-
ropsy and collection of tissues for histopathological
examination. At the end of the study, the remaining ani-
mals were killed. In additional studies two groups of 20
rats each received 0.14 or 1.4 g/kg/day for 18 months
and then were killed for necropsy. The mixture did not
induce significant pathologic changes at any of the dose
levels. At the highest dose, a significant decrease in body
weight gain was noted during months 4–8. At lower
doses, some evidence of growth stimulation was noted.
 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
In a subcutaneous injection study, Mason et al.
(1971) administered methyl paraben at doses of 0.6,
1.1, 2.0, 3.5 mg/kg to groups of 20, 40, 60 and 80 Fischer
rats, respectively, twice weekly for 52 weeks. At the end
of the study, some animals were killed and others were
observed for an additional 6 months and then killed
for necropsy. Toxicity of methyl paraben was deter-
mined by survival time, weight changes and histopatho-
logical changes. Compared to controls, methyl paraben
treated rats had no significant differences in mortality,
weight gain or pathological lesions.
As cited in the SCOGS (1972) report, Sokol (1952)
fed propyl paraben in diet to rats for an 18-month per-
iod at 150 mg/kg/day. No ill effects and possibly some
growth stimulation were noted. At 1500 mg/kg/day,
growth depression was noted without any pathologic
changes. The details of this study were not available.
2.2.5.2. Carcinogenicity studies. In a series of studies, Ito
and his colleagues (Ito et al., 1986; Hirose et al., 1986;
Ito and Hirose, 1987) investigated the proliferative ef-
fects of propyl paraben in hamsters. No treatment re-
lated microscopic changes in the stomach or increases
in liver weight were seen in 15 hamsters fed a diet con-
taining 3% propyl paraben (approximately 3 g/kg body
weight/day) for 20 weeks. No histopathological lesions
were observed in the urinary bladder epithelium. Inter-
estingly, propyl paraben increased the labeling index
(p < 0.05), suggesting a proliferative response in the
bladder epithelium. Of the fifteen hamsters fed 3% pro-
pyl paraben, five animals showed mild hyperplasia in the
forestomach.
In another study, Rodrigues et al. (1986) investigated
effects of feeding 4% methyl or propyl paraben to rats
for 9 days on the [3H] thymidine labeling index and
the histological changes of the forestomach. Methyl
paraben feeding did not affect the labeling index in the
prefundic and mid-region of the rat forestomach. Simi-
larly, histopathological observations did not show
mucosal changes after methyl paraben feeding. Propyl
paraben effectively increased the labeling index in the
prefundic area of the forestomach epithelium. Similar
to the effects of methyl paraben noted by Rodrigues
et al. (1986), Clayson et al. (1986) reported that feeding
4% methyl paraben in the diet to 8 Fischer 344 male rats
for nine days did not affect the [3H] thymidine labeling
index of the rat forestomach.
In another study, Nera et al. (1984) also found that
feeding of 1% and 4% propyl paraben to rats increases
the labeling index in prefundic areas. These investigators
also noted that histologically, the degree of proliferation
in rats maintained on 4% propyl paraben for nine days
was similar to that observed with 0.5% butylated
hydroxyanisole. The histological findings included for-
mation of ‘‘rete pegs’’ and papilla, slight acanthosis,
minimal hyperkeratosis and the presence of inter-cellu-
997
lar edema. Lymphocytic cells were also seen in the lam-
ina propria. Autoradiography of the labeled cells also
revealed 2.5-fold increase in the labeling index in both
propyl paraben and butylated hydroxyanisole fed rats.
Propyl paraben, at a lower dose level (1%), also led to
a significant increase (1.5-fold) in the labeling index,
but the only histological change observed was hyperpla-
sia of the basal cells.
Kirschstein et al. (1973) studied toxic and carcino-
genic potentials of preservatives, including methyl para-
ben. Groups of mice and rats were injected (intra-venous
and subcutaneous) with methyl paraben either as single
or multiple doses. The results of the studies in mice, both
newborn and weanling, did not indicate that a signifi-
cant number of tumors occurred in test animals as com-
pared to controls.
Mason et al. (1971) also studied the carcinogenic po-
tential of methyl paraben. Some of the results from this
study were reported by Kirschstein (1973), as described
earlier in this section. Methyl paraben was subcutane-
ously injected at doses of 0.6, 1.1, 2.0, 3.5 mg/kg to groups
of 20, 40, 60, and 80 Fischer rats, respectively, twice
weekly for 52 weeks. Positive, negative and vehicle con-
trols were used. All animals were necropsied after they
died or were killed 26 weeks post-treatment. Of all tumors
observed in methyl paraben-treated rats, only mammary
fibroadenoma incidence was significantly higher than
negative control groups (8% incidence for methyl para-
ben; 1% for negative control). The incidence of injection
site tumors, pituitary adenomas, uterine polyps, and leu-
kemias did not differ significantly from controls.
In a study on carcinogenic potential of methyl para-
ben in rats and mice by different routes, methyl paraben
was found to be non-carcinogenic (Homberger, 1968).
In this study, various techniques such as subcutaneous
injection/secondary host transfer, intra-venous injec-
tion/observation of lung adenomas and cocarcinomas
for ascertaining carcinogenicity of methyl paraben were
employed. In another study, ethyl paraben fed in the
diet to 65 rats at a level of 2% (about 1 g/kg body
weight/day) for life did not show any evidence of carcin-
ogenicity (Homberger, 1968).
The above-described findings on increases in labeling
index or hyperplasia, raise important questions regard-
ing the relevance of observations in rats to humans. It
should be noted that the rat lesions described are from
the prefundic forestomach and humans do not posses
a prefundic forestomach. Additionally, and contrary to
the observations of Nera et al. (1984), Shibata et al.
(1990) did not find any increase in labeling index of
the prefundic areas in rats after propyl paraben feeding.
In the Shibata et al. (1990) study, propyl paraben was
fed to rats in the diet at 3% level for 8 weeks and alter-
ations in forestomach and glandular stomach epithelium
were evaluated. Propyl paraben did not induce any
changes or increase in labeling index in the stomach
998 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
mucosa. In the Nera et al. (1984) study, young 3–4-
week-old Fischer 344 rats were fed for nine days only,
whereas in the Shibata et al. (1990) study, adult 6
week-old Fischer 344 rats were fed for 8 weeks.
In contrast to the observations of increased labeling
index of urinary bladder in hamsters fed propyl para-
ben (Hirose et al., 1986), no significant increase in label-
ing index in the urinary bladder of Fischer 344 rats fed
propyl paraben was noted (Kurata et al., 1990). In the
Kurata et al. (1990) study, rats were fed 3% propyl
paraben for 4 weeks and the labeling index of the uri-
nary bladder was determined by bromodeoxyuridine
incorporation. Differences in these two experiments
may be due to different species and duration of propyl
paraben feeding i.e., hamster vs. rat and 20 vs. 4 weeks,
respectively.
In a screening assay for hepatocarcinogenesis, Tate-
matsu et al. (1987) investigated the role of propyl
paraben in the induction of placental glutathione S-
transferase (PGST) as a marker for hepatocarcinogene-
sis. Feeding of propyl paraben at a level of 50,000 ppm
(about 2.5 g/kg body weight/day) in diet for 8 weeks to
rats pre-treated with diethylnitrosamine, a well-estab-
lished liver carcinogen, did not result in any significant
change in PGST positive foci. In another similar study,
Ito et al. (1988) also evaluated the hepatocarcinogenic
potential of propyl paraben. Propyl paraben given in
the diet to a group of 17 rats at 50,000 ppm (about
2.5 g/kg body weight/day) for 6 weeks did not potentiate
the carcinogenic action of diethylnitrosamine. The end
point monitored was induction of pre-neoplastic PGST
positive foci.
Kurata et al. (1990) investigated the urinary bladder
tumor-promoting activity of propyl paraben in male
Fischer 344 rats. A group of animals was given 0.05%
N-butyl-N-(4-hydroxybutyl) nitrosamine as an initiator
through drinking water for 4 weeks. Three days after
the end of treatment with the nitrosamine, groups of
20 rats received a diet containing 3% (about 1.5 g/kg
body weight/day) propyl paraben or basal diet alone
until the end of week 36. At the end of 36 weeks, all ani-
mals were killed for histological examination. No signif-
icant increase in the incidence of papillary or nodular
hyperplasia, papilloma and carcinoma was noted. Feed-
ing of propyl paraben alone for 36 weeks to 10 rats at
3% level did not result in any increase in papillary or
nodular hyperplasia.
As part of a chronic toxicity study, Matthews et al.
(1956) (see Section 2.2.5.1) also investigated carcino-
genic activity of parabens in a 96-week study. Rats
maintained on diet containing 2% and 8% propyl para-
ben in the diet for 96 weeks did not show any increase in
tumors. In this study, only 6 major organs (liver, kidney,
heart, lung spleen and pancreas) were examined micro-
scopically. In another study, there was no evidence of
carcinogenicity in groups of 20 rats given 3:2 mixture
of sodium salts of propyl:ethyl paraben in the diet for
18 months at doses of 150 and 1500 mg/kg body
weight/day. Only the major organs were examined
microscopically (Applied Research Laboratories,
1942). Details of the study were not available.
In a chronic tumorigenicity study, groups of IRC/Jcl
mice (50/sex/group) were administered 0, 0.15, 0.3 and
0.6% butyl and isobutyl paraben in the feed for 102
weeks (Inai et al., 1985). Body weights and feed con-
sumption were recorded during the study period. Tu-
mors were observed at various sites including the
hematopoietic system, the lung and the soft tissue. At
none of the sites did the tumor incidence or the time
to death with tumors differ significantly from that in
the untreated control group.
In a poorly documented study, propyl paraben was
evaluated for carcinogenicity with a transplacental assay
and a newborn assay. In the transplacental assay, preg-
nant rodents were given orally, the maximum dose not
causing abortion or early death of neonates. Animals
were treated every other day for five days during days
15–19 of gestation. Sucklings were observed for one year
after birth for tumor development. In the newborn
study, four subcutaneous injections of propyl paraben
(total dose = LD20) were administered to rodent pups
on days 1, 8, 15 and 22 following birth. Sucklings were
observed for one year after birth for tumor develop-
ment. In both studies, propyl paraben was reported to
be non-carcinogenic (Odashima, 1976).
2.2.6. Teratogenicity/reproduction toxicity
In an extensive study, Food and Drug Research Lab-
oratories performed teratologic studies of methyl para-
ben in mice, rats, hamsters (FDRL, 1972) and rabbits
(FDRL, 1973). In these studies, administration of up
to 550 mg/kg (maximum dose studied) of methyl para-
ben to pregnant mice or rats for 10 consecutive days
(from Day 6 of gestation to Day 15) and up to
300 mg/kg (maximum dose studied) to hamsters (5 days;
from Day 6 of gestation to Day 10) or rabbits (13 days;
from Day 6 through Day 18) had no clearly discernible
effect on nidation or on maternal or fetal survival. The
number of abnormalities seen in either soft or skeletal
tissues of the test groups did not differ from the number
occurring spontaneously in the sham-treated controls.
These studies demonstrate that methyl paraben is not
embryotoxic or teratogenic in rats, mice, hamsters and
rabbits (FDRL, 1973). Similarly, ethyl paraben, given
to rats at 0.1% and 10% through diet for one week
during gestation days 8–15, did not show any teratoge-
nicity or toxicity. The authors of this study concluded
that ethyl paraben, at concentrations up to 10% in the
diet, was not teratogenic (Moriyama et al., 1975;
BIBRA, 1989a,b). The reproductive toxicity studies of
 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
parabens are presented below in the section on estro-
genic activity.
2.2.7. Genotoxicity studies
In two separate reports, Litton Bionetics (1975, 1974)
investigated the mutagenic potentials of methyl and pro-
pyl paraben by Ames test using Salmonella typhimurium
strains TA100, TA98, TA1535 and TA1537. Assays
were performed with and without Aroclor 1254-induced
rat liver S-9 fraction. Neither methyl nor propyl paraben
exhibited mutagenic activity in any of the in vitro assays
employed (Litton Bionetics, 1975; McCann et al., 1975;
Morita et al., 1981). When tested at doses of 10–2000 lg/
plate, propyl paraben was non-mutagenic in the pres-
ence and absence of metabolic activation (McCann
et al., 1975). In a modified Ames assay, Sugimura
et al. (1976) added propyl paraben in dimethyl sulfoxide
to cultures of S. typhimurium strains TA100 and TA98
and to E. coli strain D-2. Experiments were conducted
with and without polychlorinated biphenyls induced
rat liver microsomal S-9 fraction. Except in strain
TA100 under metabolic activation, propyl paraben
was non-mutagenic in all other strains. A rec assay in
the bacterium Bacillus subtilis, an indirect measure of
DNA damage, gave negative results both in the presence
and absence of liver metabolic activation (Morita et al.,
1981). A brief citation implies that a similar rec assay in
the bacterium E. coli gave positive results (Odashima,
1976).
In three different mutagenicity assays, Litton Bionet-
ics (1974) evaluated the mutagenic potentials of methyl
paraben by a host-mediated assay, a cytogenetic assay,
and a dominant lethal assay. The host-mediated assay
consisted of three parts, an acute in vivo test, a sub-
chronic test, and an in vitro study. In the acute test
0–5000 mg/kg methyl paraben was administered orally
to each of 10 mice. Positive and negative controls were
used. After the treatment with methyl paraben, animals
were given intra-peritoneally, 2 ml S. typhimurium strain
TA 1530 and 2 ml of Saccharomyces cerevisiae strain D-
3 indicator organisms. All animals were killed 3 h later
and peritoneal fluid was extracted, bacterial counts were
made, and the numbers of mutations were recorded.
Methyl paraben induced no significant increases in mu-
tants or recombination frequencies. In the subchronic
test, each of 10 mice received orally, 0–3500 mg/kg
methyl paraben daily for five consecutive days. Within
30 min after the last treatment, animals were inoculated
with indicator organisms, S. typhimurium strain TA
1530 and S. cerevisiae strain D-3 and treated as de-
scribed above for the intra-peritoneal tests. No signifi-
cant increases in mutants or recombination frequencies
were noted. In the in vitro study, 0–100 lg/ml methyl
paraben was added to plates containing S. typhimurium
strain TA 1530 and S. cerevisiae strain. After incuba-
999
tion, the number of mutants was recorded. In the
in vitro study, methyl paraben induced no significant in-
creases in mutant or recombinant frequencies with
S. typhimurium or S. cerevisiae.
In the cytogenetic assay, administration of a single or
multiple (daily for 5 days) doses of methyl paraben at
levels of 0–5000 mg/kg to rats did not induce detectable
aberrations in the chromosomes of the rat bone marrow
cells in metaphase. Similarly, in an in vitro study, methyl
paraben did not induce any significant aberrations in the
anaphase of cultures of human embryonic lung cells. In
rats, fewer mitoses were observed in bone marrow cells
of rats treated 5000 mg/kg/day for five days. The inves-
tigators of this study suggested that methyl paraben
might interfere with mitosis when administered ‘‘subch-
ronically’’ at high doses. In the dominant lethal test,
male rats were treated orally with 0–5000 mg/kg methyl
paraben once (acute study) or daily for 5 days (sub-
chronic study). After treatment with methyl paraben,
males were mated with two virgin females per week
for 7–8 weeks. On Day 14, pregnant rats were killed
and uteri were examined for deciduomata, late fetal
deaths, and total implantations. No-dose response or
time-trend patterns were observed that would suggest
a dominant lethal effect for methyl paraben. Methyl
paraben was non-mutagenic under the conditions of
the study (Litton Bionetics, 1974).
In an article in Japanese, Ishidate et al. (1978) re-
ported on the clastogenicity of methyl-, ethyl-, propyl-
and butyl-paraben by chromosome aberration tests in
Chinese hamster cells. Aberrations studied included
chromatid breaks, chromatid gaps, chromosomal ex-
changes and ring formations. Individual parabens, at
various doses, were applied directly to cells and 24–
48 h after the application; chromosome preparations
were made to study potential aberrations. The maxi-
mum tolerated dose for methyl-, ethyl-, propyl- and
butyl-paraben were 0.5, 0.25, 0.125 and 0.06 mg/ml,
respectively. All parabens, except methyl paraben, in-
duced 1–3% increases in polyploid cells. As the paraben
alkyl chain length increased, the frequency of polyploid
cell production increased. Among the parabens tested,
ethyl and methyl paraben were found to induce a signif-
icant number of chromosomal aberrations (11% and
15% increases, respectively, over control).
In another study, Matsuoka et al. (1979) studied the
in vitro chromosomal aberration potentials of methyl
paraben (0.125 mg/ml) in Chinese hamster lung cells in
the presence and absence of polychlorinated biphenyl-
induced rat liver microsomes (S-9 mix). In the absence
of S-9 mix, no induction of chromosomal aberration
was noted. However, in the presence of S-9 mix, aberra-
tion incidence increased to 13% and was judged to be
significant. Gaps, breaks, exchange and rings were ob-
served. Contrary to these in vitro studies, results of
1000 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
in vivo investigations show that methyl paraben is non-
mutagenic.
2.2.8. Cytotoxicity studies
Nakagawa and Moldeus (1998) investigated the role
of metabolism on cytotoxic action of parabens in freshly
isolated hepatocytes. Incubation of hepatocytes with
propyl paraben at concentrations of 0.2–2.0 mM elicited
a concentration and time dependent cell death. The pro-
pyl paraben-induced cytotoxicity was enhanced when
metabolism of propyl paraben to p-hydroxybenzoic acid
was inhibited by the carboxylesterase inhibitor, diaz-
inon. In a comparison of toxic potency, butyl and iso-
butyl paraben were more toxic than propyl- and
isopropyl paraben. While, ethyl and methyl paraben
and p-hydroxybenzoic acid were less toxic than propyl
paraben. Propyl paraben caused a concentration and
time dependent decrease in the levels of ATP and gluta-
thione, which was accompanied by loss of cell viability.
These in vitro studies indicate that propyl paraben itself,
and not products of its hydrolysis, are the cytotoxic
compounds. In in vivo conditions, carboxyesterase en-
zymes rapidly hydrolyze propyl paraben.
The mechanism of cytotoxicity of parabens is not
clear. Incubation of propyl paraben with Krebs–Hanse-
leit buffer did not elicit an increase in oxygen consump-
tion indicating that autooxidation via formation of
superoxide anions radicals does not occur readily
(Nakagawa and Moldeus, 1992, 1998). In a hepatocyte
suspension, propyl paraben did not result in accumula-
tion of malondialdehyde, an indicator of lipid peroxida-
tion (Nakagawa and Moldeus, 1998). These studies
suggest that formation of free radicals, a major mecha-
nism in the onset of lipid peroxidation (Fawthrop
et al., 1991), is unlikely to be involved in the cytotoxic-
ity. The toxic potency of parabens to hepatocytes
or mitochondria depends on the relative elongation of
alkyl side-chains esterified to the carboxyl group of
p-hydroxybenzoic acid.
Studies by Nakagawa and Moldeus (1998) suggest
that propyl paraben causes uncoupling of oxidative
phosphorylation, inhibits NAD+ and FAD linked mito-
chondrial respiration and reduces mitochondrial mem-
brane potential. Impairment of mitochondrial function
is known to result in a decrease in the rate of cellular
ATP synthesis and thus the nucleotide level. Mitochon-
drial dysfunction and its consequences are common
mechanisms of cytotoxicity for a wide range of chemicals
and a number of pathological conditions (Kehrer et al.,
1990). Thus it appears that propyl paraben-induced cyto-
toxicity is mediated by mitochondrial dysfunction.
In another study, Nakagawa and Moore (1999)
investigated the relationship between mitochondrial
membrane permeability transition and the toxic effects
of parabens in mitochondria and hepatocytes isolated
from rat liver. In isolated mitochondria, propyl para-
ben at a concentration of 0.1–0.5 mM, in the presence
of 50 mM Ca2+, caused a dose-dependent induction
of mitochondrial swelling. For the other parabens
(methyl, ethyl and butyl) tested, the induction of mito-
chondrial membrane permeability transition depended
on the relative elongation of alkyl side-chains in their
molecular structure and was associated with the parti-
tion coefficients. Pre-treatment with cyclosporin A, spe-
cific inhibitor of membrane permeability transition,
prevented the swelling. In freshly isolated hepatocytes,
cyclosporin A and trifluoperazine, which inhibit mem-
brane permeability transition in a synergistic manner,
partially prevented propyl-paraben plus diazinon (an
inhibitor of esterases involved in the breakdown of pro-
pyl paraben) induced cell death, ATP loss and de-
creased mitochondrial membrane potential. These
studies suggest that the onset of paraben-induced cyto-
toxicity is linked to mitochondrial failure dependent
upon induction of membrane permeability transition
accompanied by the mitochondrial depolarization and
depletion of cellular ATP through uncoupling of oxida-
tive phosphorylation.
In embryonic mouse fibroblast cultures, methyl-,
ethyl- and propyl-paraben significantly reduced biosyn-
thesis of both RNA and DNA. Interestingly, none of
the parabens affected the protein content of the cell cul-
tures (Krauze and Fitak, 1971). Sheu et al. (1975) re-
ported that the IC50 (dose for 50% cell inhibition) of
propyl paraben in HeLa cells was 0.22 mM. In another
in vitro study, methyl and propyl paraben, at concentra-
tions of 0.25% and 0.05%, respectively, did not induce
hemolysis of human and rabbit erythrocytes (Ansel
and Cadwallader, 1964). Nes and Eklund (1983) studied
the effects of parabens, including methyl paraben on
DNA, RNA, and protein synthesis in E. coli and B. sub-
tilis. Both DNA and RNA synthesis in cells permeabili-
zed by toluene in E. coli and B. subtilis were inhibited.
Protein synthesis in cell free extracts (S-30 fraction) of
B. subtilis was even more sensitive to parabens than
DNA and RNA synthesis, while protein synthesis in
E. coli was largely unaffected. Among the parabens
tested, methyl paraben was least sensitive.
In an in vitro test to assess the release of lysosomal
enzymes from peripheral human phytohaemaglutinin
stimulated lymphocytes in culture, methyl-, ethyl-, pro-
pyl- and butyl-parabens caused a concentration-depen-
dent diminution of the secretion of lysosomal enzymes
(Bairati et al., 1994). Butyl paraben caused 45–50% inhi-
bition at 0.06 mmol/l and appeared to be the most po-
tent inhibitor. For other parabens, the inhibitory effect
became statistically significant at about 0.25 mmol/l
for alpha-L-fucosidase and alpha-D-galactosidase, and
at 0.5 mmol/l for N-acetyl-beta-D-glucosaminidase and
beta-D-glucuronidase. For all the enzymes, at a concen-
tration of 1 mmol/l, inhibition was greater than 50% and
was more marked with the propyl paraben. In this
 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
study, parabens did not influence the release of LDH,
suggesting that they affected particularly the secretion
of lysosomal enzymes. The investigators suggested that
parabens are capable of affecting cellular function at
concentrations that are likely to be reached in blood
or tissues under conditions of common use. However,
under in vivo conditions, methyl paraben is known to
be metabolized rapidly.
In an in vitro study, Bao-Liang et al. (1989) tested
spermicidal activity of methyl-, ethyl-, propyl- and
butyl-paraben. The effects of parabens on spermicidal
activity was assessed in sperm samples from 19 human
subjects by the Agreed Test of Total Spermicidal Power
proposed by Harris (1962) and accepted by the Interna-
tional Planned Parenthood Federation. The spermicidal
potency or pass point concentration of methyl, ethyl-,
propyl- and butyl paraben was found to be 6, 8, 3 and
1 mg/ml. These studies demonstrate that parabens are
an effective spermicide for human spermatozoa.
Batts et al. (1990) studied the effects of methyl and
propyl paraben on nasal cell ciliary beat frequency using
a photoelectronic technique. Both the parabens inhib-
ited beat frequency, an effect that was reversible by rins-
ing. These studies indicate that direct application of
parabens exhibited reversible toxicity to cilia in the ab-
sence of mucus. Mostow et al. (1979) studied the effects
of methyl and propyl paraben on the ciliary activity of
epithelial cells, in culture (harvested from ferret tracheal
rings). Both methyl and propyl paraben inhibited ciliary
activity. The results of this study indicate that topical
respiratory anesthesia with paraben-containing solu-
tions may result in prolonged ciliary paralysis. In a re-
view article on the toxicological implication of nasal
formulations, Quadir et al. (1999) reported that methyl
and propyl paraben inhibits ciliary beat frequency in
frogs in a concentration-dependent manner, but the ef-
fect is reversible.
Jian and Li Wan Po (1993a,b) reported mild ciliotox-
icity of propyl paraben at concentrations of 0.28 and
0.38 mM in rat trachea. These investigators also re-
ported that propyl paraben and methyl paraben to-
gether exert obvious synergism in their ciliotoxicity.
The ciliotoxicity of 10% methyl paraben was more pro-
nounced in the presence of 30% propyl paraben than in
its absence. Additive effects of methyl paraben and pro-
pyl paraben could not explain the increased ciliotoxicity.
No synergism was apparent when methyl paraben con-
centration was increased from 10% to 20% in the pres-
ence of 30–40% propyl paraben. Interestingly, the
synergistic effects of methyl paraben and propyl paraben
can only be observed over a certain concentration range
of each chemical.
2.2.9. Estrogenic activity
In recent years, several in vitro andin vivo studies on
the estrogenic activity of parabens have been published.
1001
These studies have generated a debate on the role of
parabens in the increased incidence of breast cancer
and reproductive toxicity. These studies (in vitro and
animal) are further discussed below, while relevance of
these studies to humans, along with other studies, are
presented in section on observations in human.
2.2.9.1. In vitro studies. Routledge et al. (1998) studied
the estrogenic activity of methyl-, ethyl-, propyl- and
butyl-paraben in a yeast-based estrogen assay. All para-
bens were found to be weakly estrogenic with the
most potent (butyl paraben) being 10,000-fold less
potent than 17b-estradiol. The magnitude of the estro-
genic response increased with alkyl group size and
methyl-, ethyl- and propyl-paraben was approximately
2,500,000-fold, 150,000-fold and 30,000-fold less potent
that 17b-estradiol, respectively. Interestingly, the pri-
mary metabolite of the parabens, p-hydroxybenzoic
acid, was found inactive. 4-Hydroxytamoxifen inhibited
the in vitro estrogenic activity of parabens, suggesting
that parabens interact with the estrogen receptor. In this
assay, parabens possess estrogenic activity with poten-
cies between 104- and 107-fold lower than that of 17b-
estradiol. Similarly, in more recent studies, Miller
et al. (2001) and Vinggaard et al. (2000) noted weak
estrogenic activity paraben in the yeast assay. Okubo
et al. (2001) also reported estrogenic activity of ethyl-,
propyl-, butyl-, isopropyl- and isobutyl paraben in hu-
man MCF-7 breast cancer cells. Similarly, Darbre et
al. (2002, 2003) reported estrogenic activity of isobutyl
paraben and benzyl paraben in human MCF-7 and
ZR-75-1 cell lines.
In another study, Blair et al. (2000) investigated
estrogen receptor competitive binding affinities of para-
bens (methyl, ethyl, propyl, butyl, benzyl, heptyl and 2-
ethylhexyl). Hysterectomized Sprague–Dawley rats were
the estrogen receptor source for the competitive binding
assay. Test chemicals that exhibited affinity for the
estrogen receptor in the first tier were subsequently as-
sayed using a wide range of concentrations to character-
ize the binding affinity. All of the parabens examined in
this study competed for estrogen receptor. The parabens
demonstrated a positive correlation between binding
affinity and chain length. Increase in chain length
showed increase in relative binding affinity.
Okubo et al. (2001) examined estrogenic activities of
methyl-, ethyl-, propyl-, butyl-, isopropyl- and isobutyl-
paraben by assaying estrogen-receptor dependent prolif-
eration of MCF-7 cells. All the parabens stimulated the
proliferation to about the same level as the maximal cell
yield attained with 3 · 10 11 M 17b-estradiol, but at a
concentration in the order of 105–107 higher than 17b-
estradiol. In vitro competitive binding to human estro-
gen-receptor a and b, revealed that the parabens with
longer side-chains showed greater affinity for estrogen
receptors, and that they had similar relative binding
1002 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
affinity values to both estrogen-receptor a and b. The
investigators concluded that parabens have estrogen
receptor-dependent estrogenic activities, and their effects
on the intra-cellular signaling pathway might be differ-
ent from that of 17b-estradiol. These investigators also
reported that in addition to estrogenic activity, butyl
and isobutyl paraben increased progesterone receptor
gene expression.
2.2.9.2. In vivo studies. Hossaini et al. (2000) studied the
estrogenic activity of methyl-, ethyl- and propyl-paraben
in a mouse uterotrophic assay. Immature B6D2F1 mice
(a strain used to screen compounds with estrogenic
activity) were treated with oral or subcutaneous doses
of the paraben test compounds for three consecutive
days. Additionally, p-hydroxybenzoic acid and butyl
paraben were tested by the subcutaneous route in a rat
uterotrophic assay. A significant increase in uterus
weight on Day 4 was considered an estrogenic effect.
In the mouse assay, none of the parabens produced
any estrogenic response at dose levels up to 100 mg/kg/
day. For ethyl paraben even at a dose level of
1000 mg/kg/day, no estrogenic activity was noted. Sub-
cutaneous administration of butyl paraben to immature
Wistar rats produced a weak estrogenic response at
600 mg/kg/day. In another study, subcutaneous admin-
istration of isobutyl paraben was able to increase the
uterine weight in the immature mouse after three daily
doses of 1.2 or 12 mg per mouse (Darbre et al., 2002).
In another study, ethyl-, propyl- and butyl paraben
and their common metabolite p-hydroxybenzoic acid,
were investigated for their ability to evoke an estrogenic
response in sexually immature rainbow trout (Pedersen
et al., 2000). Induction of yolk protein was used as an
estrogen-specific endpoint after repeated injections of
the test material. All parabens, at a dose of 100–
300 mg/kg/day were estrogenic, while the metabolite
(i.e., p-hydroxybenzoic acid), did not show any estro-
genic activity. Ethyl paraben was found to be about
one-sixtieth (1/60), the potency of propyl- or butyl para-
ben which had estrogenic potencies comparable to
bisphenol A (Pedersen et al., 2000).
Lemini et al. (1997) investigated the estrogenic activ-
ity of p-hydroxybenzoic acid (the primary metabolite of
paraben) in immature and adult ovariectomized female
mice (CD1) using two bioassays. Subcutaneous adminis-
tration of different doses of p-hydroxybenzoic acid were
compared with estradiol (E2), and their effects on vagi-
nal cornification and uterotrophic activities were evalu-
ated. Animals were treated subcutaneously with 0, 0.5,
5, 50 and 500 lg/100 g daily for 3 days or E2 (1 lg/
100 g). Four days after treatment, p-hydroxybenzoic
acid produced a dose-dependent response on vaginal
cornification and uterotrophic activity in both immature
and adult ovariectomized mice. The relative utero-
trophic potency of p-hydroxybenzoic acid (500 lg/
100 g) to E2 (1 lg/100) was 0.0011 in immature mice
and 0.0018 in ovariectomized animals.
In a recent study, Kang et al. (2002) investigated the
effects of maternal exposure of butyl paraben during
gestation and lactation periods on the development of
the reproductive organs of the F1 offspring. Time-mated
pregnant Sprague–Dawley rats were injected subcutane-
ously with 100 or 200 mg/kg of butyl paraben on gesta-
tion day 6 to post-natal day 20. In the group treated
with 200 mg/kg of butyl paraben, the proportion of pups
born alive and surviving to weaning were decreased. At
post-natal day 49, the body weights of female offspring
were significantly decreased. Effects in male F1 offspring
varied as function of dose level and post-natal time and
no apparent pattern emerged. At post-natal day 49,
weights of testes, seminal vesicles and prostate glands
were significantly decreased in rats exposed to 100 mg/
kg of butyl paraben. Butyl paraben administration did
not affect the weights of female reproductive organs in
pups. In both the groups, the sperm count and the sperm
motility in the epididymis were significantly decreased.
In accordance with the sperm count in the epididymis,
the number of round spermatids and elongated spermat-
ids in the seminiferous tubule (stage VII) were signifi-
cantly decreased by butyl paraben. At post-natal day
90, testicular expression of a- and b-estrogen receptor
mRNA was significantly increased in the group treated
with 200 mg/kg of butyl paraben. These results indicate
that maternal exposure to butyl paraben may adversely
affect the F1 male offspring.
In a series of studies in rodents, Oishi (2001, 2002a,b,
2004) investigated male reproductive effects of parabens.
In the first study, groups of male Wistar rats (n = 8)
were fed diet containing 0%, 0.01%, 0.1% and 1% butyl
paraben for eight weeks. At the end of the treatment, the
rats were killed and the weights of the testes, epididymi-
des, prostates, seminal vesicles and preputial glands
were recorded. A dose related decrease in the absolute
and relative weights of epididymides and serum testo-
sterone concentration was noted and for both the
parameters the decreases were statistically significant
at 0.1% and above. A significant decrease in the cauda
epididymal sperm reserve was noted in all the treated
groups. The sperm count of the group receiving the
highest dose, was 58.2% of control values. Compared
to controls, the daily sperm production in the testis
was also significantly lower in all treated groups. The
authors claimed that the daily intake of butyl paraben
that caused these disruptions, is similar to acceptable
daily intake (ADI) levels for parabens in the European
Community and in Japan (Oishi, 2001).
In the second study, Oishi (2002b) fed groups of male
Crj:CD-1 mice (n = 8) diets containing 0%, 0.01%, 0.1%
and 1% (0, 14, 146, 1504 mg/kg/day) butyl paraben for
10 weeks. No treatment-related effects of butyl paraben
on the liver, ventral prostates, seminal vesicles and pre-
 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
putial glands were noted. In animals receiving 1% butyl
paraben, a significant increase in both the absolute and
relative weights of the epididymides was noted. A dose-
related decrease of both round and elongated spermatid
counts in stages VII–VIII seminiferous tubules were
noted. All the groups receiving butyl paraben showed
a significant decrease in the elongated spermatid counts.
Compared to control, the numbers of spermatogonia
and spermatocytes did not differ. A dose-dependent de-
crease in the serum testosterone concentration was
noted and the decrease was significant at 1% of butyl
paraben.
In the third study, Oishi (2002a) fed groups of male
rats (n = 8) with AIN93G modified diet containing 0%,
0.01%, 0.1% and 1% propyl paraben for four weeks.
At the end of the treatment, the rats were killed and
the weights of testes, epididymides, prostates, seminal
vesicles and preputial glands were recorded. No treat-
ment-related effects of propyl paraben on the organ
weights in any of the study groups were noted. A
dose-related decrease in the cauda epididymal sperm re-
serves and concentrations was noted and the difference
was significant at dose of 0.1% and above. A significant
decrease in daily sperm production and its efficiency in
the testis of all groups receiving propyl paraben were
reported. A dose related decrease in the serum testoster-
one concentration was noted. The decrease was signifi-
cant in the group that received the highest dose.
In a recent study, Oishi (2004) has shown that methyl
and ethyl parabens do not adversely affect the secretion
of sex hormones or the male reproductive function. In
this study, methyl and ethyl parabens were fed at levels
of 0.1% and 1.0% each in the diet to five groups of 25–
27-day-old rats (8/group). At the end of eight weeks, the
rats were killed and the weights of the testes, epididymi-
des, prostates, seminal vesicles and preputial glands
were determined. There were no treatment-related ef-
fects of either compound on the organ weights in any
of the study groups. Neither compound exhibited anti-
spermatogenic effects nor elicited changes in levels of
testosterone, LH and FSH at a dose level of about
1000 mg/kg of body weight per day. The results of this
and previously described studies suggest that increase
in alkyl chain length increases the estrogenic activity
of parabens.
2.2.10. Neurotoxicity
In a study on the toxicity of local anesthetic additives,
including parabens, Rowlingson (1993), concluded
methyl and propyl parabens did not show any neuro-
toxic effects. In animal studies, when large doses were in-
jected intra-thecally or perineurally, methyl paraben did
not produce long-lasting neural blockade or histological
evidence of damage in rabbits (Adams et al., 1977;
Mizuno et al., 1994). Systemic or in vitro application
of methyl and propyl paraben dilated cerebral blood
1003
vessels (Brandt et al., 1983; Hamilton et al., 1990). How-
ever, Cerda et al. (1997) reported intra-thecal injection
of amitryptyline plus methyl and propyl paraben, at
high doses did not alter the spinal cord blood flow in
conscious sheep, nor did it produce behavioral signs of
neurotoxicity after a single dose.
2.2.11. Antimicrobial activity
As parabens are effective against fungi and bacteria at
low concentrations, they are used extensively in pharma-
ceutical formulations as preservative agents. The antimi-
crobial activity of parabens is higher against fungi
compared with bacteria. Parabens are more active
against Gram-positive bacteria than Gram-negative bac-
teria. A summary of antifungal and antibacterial prop-
erties of parabens against a number of microbial
strains is presented in the Final Report on the Safety
Assessment of Methyl Paraben, Ethyl Paraben, Propyl
Paraben and Butyl Paraben (Elder, 1984). Generally,
parabens are effective in acid, neutral and slightly alka-
line solutions. However, above pH 8, hydrolysis of para-
ben may occur and can reduce the preservative efficiency
(Aalto et al., 1953; Rosen and Berke, 1973). Parabens
act on both the germinative and vegetative phases of
microbial development. Spore germination is much
more susceptible to parabens than vegetative growth in
fungi or bacteria (Bomar, 1962; Watanabe and Takesue,
1976). Because of their claimed synergistic effects, com-
binations of methyl paraben and propyl paraben are
often added to aqueous formulations (Berenbaum,
1977). However, Gilliland et al. (1992) demonstrated
that these two parabens exert additive rather than syner-
gistic antimicrobial effects.
The presence of a co-solvent also affects the effective-
ness of parabens. Darwish and Bloomfiled (1997) re-
ported that the increase in antibacterial activity of
methyl and propyl paraben preservatives, in co-solvent
solution with ethanol, propylene glycol and glycerol,
can largely be accounted for by their combined effects
on the integrity of cell membranes. In an earlier study,
Darwish and Bloomfiled (1995) reported that co-solvent
concentration of 3 M increases the solubility of methyl
and propyl paraben. The increased solubility of the
parabens in the presence of co-solvent over their aque-
ous solubility was associated with increased antimicro-
bial activity. In another study, the abilities of nine
antimicrobial systems to preserve an experimental
water-based cosmetic formulation were evaluated by
six microbiological evaluation tests (Farrington et al.,
1994). The antimicrobial system contained various com-
binations and amounts of methyl and propyl paraben.
All of the tests were equivalent predictors of preserva-
tion efficacy.
Okada et al. (1999) determined minimum and 50%
growth inhibitory activity of propyl paraben for S.
cerevisiae and Klebsiella pneumoniae. The minimum
1004 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
inhibitory concentration of propyl paraben for these
two microbes was found to be 1.29 and 4.28 mM, while
the 50% inhibitory concentration was reported as 0.88
and 2.77 mM, respectively. Propyl paraben, at a concen-
tration of 200 ppm in thiotone-yeast extract-glucose
growth medium, inhibited the germination and toxin
production of C. botulinum 10755A for up to 120 h at
37 C. In another study, growth and toxin production
of C. botulinum were only delayed when 100 ppm propyl
paraben was added to the growth medium (Robach and
Pierson, 1978).
In a series of experiments, Thompson (1994) deter-
mined the minimum inhibitory concentration (MIC) of
ethyl-, methyl-, propyl- and butyl-paraben against
mycelial growth of several mycotoxigenic species of
Aspergillus, Fusarium and Penicillium. Results of this
study indicated that butyl and propyl paraben were
the most effective parabens with complete mycelial inhi-
bition at concentrations ranging from 1–2 mM for the
majority of the fungi. Of the four parabens, methyl
paraben was the least effective. The results of this study
suggest that the effect of dual combinations of parabens
in equimolar concentrations could be effective against
mycotoxigenic fungi.
Parabens can affect the growth of oral bacteria. Stein-
berg et al. (1999) reported that methyl and propyl para-
ben, at a concentration of 0.6% and 0.3% in a
mouthwash, had little effect on oral bacterial counts;
however, parabens in a slow release device had a signif-
icant and extended effect in reducing oral bacteria. Para-
bens can augment the acid sensitizing effects of weak
acids, such as fluoride for the cariogenic bacterium
Streptococcus mutans, and thus could affect the organ-
ismÕs capacity to cause dental caries (Belli et al., 1995;
Steinberg et al., 1999). Parabens irreversibly inhibited
glycolysis in the cariogenic dental plaque bacterium
Streptococcus mutans GS-5 and decreased the capacity
of the bacterium to lower the pH in dense cell suspen-
sion containing excess glucose (Ma and Marquis,
1996). The effectiveness increased with increasing chain
length. The authors reported that the lethal action of
paraben could be interpreted at least in part as due to
irreversible damage to key enzymes, such as those of
the phosphotransferase system.
In another study, Furr and Russell (1972) reported
that propyl- and butyl paraben induced leakage of
intra-cellular material through the cell wall of the bacte-
rium, Serratia marcescens. As no gross cellular damage
was noted, these agents do not appear to cause lysis.
The authors concluded that parabens act by damaging
the cytoplasmic membrane. In a subsequent study, Rus-
sell and Furr (1986) investigated the effects of different
parabens on smooth, rough and deep rough strain of
S. typhimurium. As the homologous series of parabens
ascended, activity increased (Bahgat et al., 1983; Russell
and Furr, 1986).
Shiralkar et al. reported that within 2 min after the
addition of the propyl paraben, approximately 90–95%
of the compound was taken up by the cultures of Aero-
bacter species (Shiralkar et al., 1977, 1978). The rapid
uptake of paraben by the cultures suggest that uptake
of paraben is a physical phenomenon rather than a re-
sult of active biological transport. The authors also
reported that parabens have no effect on nutrient trans-
fer into the cell or on hydrolytic enzymes. Parabens have
a significant inhibitory effect on oxygen consumption
(respiration) and most oxidative enzymes. Shiralkar
et al., 1978) stated that microorganisms may survive in
the presence of parabens if they possessed an esterase
that could hydrolyze the ester linkage of the parabens.
In another study, Close and Neilsen (1976) identified
an organism that possesses the capacity to hydrolyze
the parabens. A propyl paraben-resistant strain of Pseu-
domonas cepacia was able to use propyl paraben as a
carbon source once it was hydrolyzed. Valkova et al.
(2001) isolated Enterobacter cloacae strain EM from a
commercial dietary mineral supplement stabilized by a
mixture of methyl and propyl paraben. In liquid culture,
this strain hydrolyzes approximately 500 mg/l propyl
paraben in 2 h.
2.3. Observations in humans
2.3.1. Clinical observations
In a study of 186 patients with candidiasis (from Can-
dida albicans), McVay and Sprunt (1951) reported that
oral, vaginal or rectal administration of methyl and pro-
pyl paraben effectively inhibited development of candi-
diasis during antibiotic (aureomycin) treatment. In
three patients with candidal vaginitis, intra-vaginal
application of 200 mg paraben daily, ameliorated symp-
toms. Parabens did not cause any toxic effects in the
study subjects. In another study, Metzger et al. (1954)
administered each of the 17 patients 90 mg methyl para-
ben plus 22.5 mg propyl paraben and aureomycin, three
times daily for three days. Feces samples were examined
daily for the presence of yeast. Compared to control pa-
tients receiving aueromycin alone, paraben administra-
tion exerted antifungal activity. These studies suggest
that parabens might be useful in controlling yeast over-
growth during antibiotic treatment.
In a phase-I human safety assessment study, Eisenach
et al. (1997) investigated the effects of intra-thecal neo-
stigmine containing methyl and propyl paraben as pre-
servatives. Intra-thecal administration of neostigmine
solutions with and without preservatives produced a
dose-dependent analgesia, nausea, weakness and seda-
tion. In a study of dental alveolitis, Ritzau and Swan-
gsilpa (1975) studied the prophylactic effects of propyl
paraben. In this study, immediately following removal
of a mandibular third molar, each of the 45 patients
received three tablets containing either 33 mg propyl
 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
paraben or a placebo in the socket. None of the patients
receiving propyl paraben developed alveolitis, whereas
24% of the placebo group did. The prophylactic effect
of propyl paraben was highly significant without any
side effects of the treatment.
Gelb et al. (1992) studied changes in cerebral blood
flow before and after the intra-venous administration
of methyl and propyl paraben. Eight healthy adult sub-
jects were treated intra-venously with 9 mg methyl para-
ben plus 1 mg of propyl paraben. Cerebral blood flow
and flow velocity were measured with inhaled 133Xenon
and transcranial Doppler, respectively. No changes in
either cerebral blood flow or velocity were noted. The
investigators concluded that methyl and propyl paraben
do not cause the rise in intra-cranial pressure seen with
succinylcholine.
2.3.2. Human irritation and sensitization studies
Numerous studies on paraben irritation and sensiti-
zation have appeared in literature. In a review article,
Elder (1984) described several studies of human skin
irritation with a number of product formulations con-
taining parabens at concentrations of 0.1–0.8%. Single
insult occlusive patch tests with three formulations con-
taining paraben produced no or only minimal irritation.
In a 5-day cumulative irritancy test with a hairdressing
product containing parabens, no irritation was noted.
Daily skin patch testing of seven product formulations
for 20–21 days produced results of essentially no irrita-
tion to moderate irritation. Controlled use of two eye
makeup formulations for four weeks produced no irrita-
tion. Skin sensitization test in 3455 human subjects did
not show reactions. In this same review article, Elder
(1984) summarized sensitization results of patch tests
of different parabens on 27,230 patients with dermatitis.
Patch testing with 1–30% parabens resulted in sensitiza-
tion in approximately 2.2% of patients. Subjects with
normal (intact) skin did not develop sensitivity to para-
bens. Generally, patients with high sensitivity towards
‘‘para-agents’’ showed higher sensitivity to parabens.
Parabens (methyl, ethyl, propyl and butyl) were each
applied to the backs of 50 human subjects at concentra-
tions of 5%, 7%, 10%, 12% and 15% in propylene glycol
daily for five days. Patches were then removed and sites
scored. Propyl paraben at a concentration of up to 12%
did not produce any irritation; at the 15% concentra-
tion, it was irritating. In a repeated insult patch test
(RIPT), each paraben at the ‘‘no effect’’ concentration
above was applied to the skin of 50 subjects (25 males
and 25 females) for 4–8 h every other day for three
weeks (ten applications). Following a 3-week rest, the
materials were reapplied at induction concentrations
for 24–48 h. No sensitization was reported (Sokol,
1952).
In a repeated insult patch test, Marzulli et al. investi-
gated the sensitizing potentials of mixtures of methyl
1005
and propyl paraben in male subjects (Marzulli et al.,
1968; Marzulli and Maibach, 1973). The test mixture
was applied to the subjectsÕ arm for 48 h under occlu-
sion. Application of the test mixture was repeated every
48 h for three weeks (ten induction applications). At the
highest paraben concentration tested (10% of methyl
and propyl paraben, each), one group was alternately
irritated by topical application of 5% sodium lauryl sul-
fate under occlusion for 24 h, followed by application of
paraben for 48 h. Five such cycles were used for induc-
tion. After a 2-week rest, 72-h challenge test was per-
formed. On one test site in all subjects, 10% sodium
lauryl sulfate was applied for 1 h before challenge appli-
cation. The results of challenge test are summarized in
Table 5. The investigators concluded that sensitization
to parabens is not a problem as these parabens are used
at 0.1–0.3% in topical medicaments.
2.3.3. Photocontact sensitization
Several investigators studied the photocontact sensiti-
zation of products containing 0.2% methyl paraben and/
or 0.2% propyl paraben. In these studies, no reaction
indicative of photosensitization was noted (Elder,
1984). Similarly, no signs of photoxicity were noted in
human subjects with methyl paraben and/or propyl
paraben. Eberlein-Koenig et al. (1993) evaluated the in
vitro possible prototoxic effects of propyl paraben on
photohemolysis in suspensions of human erythrocytes.
Propyl paraben did not cause any significant photo-
hemolysis.
2.3.4. Case reports
Several reports of adverse reactions to parabens and
benzoates in sensitive individuals have been published.
As parabens and benzoates are structurally similar,
these chemicals are often considered collectively for their
sensitization reactions. Aspirin, which is structurally
related to the principal metabolite of paraben i.e.
p-hydroxybenzoate, is a well-known cause of sensitiza-
tion. Majority of the reported adverse reactions to para-
bens are relatively mild, and many of the cases involve
Table 5
Results of paraben sensitization (Marzulli et al., 1968)
Concentration in Number of individual sensitized to
petrolatum (%)a challenge
Without SLSb With SLS
a a
0.2 M + 0.05P 0/102 0/102
1.0 M + 0.25P 0/101 0/101
5.0 M + 1.25P 1/98 1/98
10.0 M + 10.0P 0/74 0/74
10.0 M + 10.0Pc 0/22 –
a
M = Methyl paraben; P = Propyl paraben.
b
Sodium laurel sulfate.
c
Sodium laurel sulfate induction phase.
1006 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
contact sensitization reactions associated with the use of
the parabens in cosmetic applications.
In a case study, Nagel et al. (1977) reported a patient
with bronchospasm and pruritis after intra-venous
administration of a hydrocortisone preparation contain-
ing methyl and propyl paraben. The patient was able to
tolerate a hydrocortisone preparation without the para-
bens. Direct and passive transfer skin tests were positive,
suggesting immediate hypersensitivity as the mecha-
nism. The sensitivity of this patient to ingested parabens
was not described.
In some cases of chronic urticaria and/or angioe-
dema, parabens have been implicated as causative fac-
tors (Ibero et al., 1982; Antico and Di Berardino,
1995). p-Hydroxybenzoic acid, a principal and common
metabolite of parabens, has also been implicated as a
causative factor in some cases of chronic urticaria and/
or angioedema (Michaelson and Juhlin, 1973; Doeglas,
1975; Thune and Granholt, 1975; Ros et al., 1976;
Warin and Smith, 1976; Gibson and Clancy, 1980;
Ehlers et al., 1998). Michaelsson and Pettersson (1974)
reported a link between ingestion of p-hydroxybenzoic
acid and exacerbation of allergic vascular purpura in
two of seven patients. Thune and Granholt (1975) inves-
tigated the effects of methyl and propyl paraben in
patients with recurrent urticaria. Thirty-seven patients
with urticaria were orally administered a tablet contain-
ing 200 mg parabens (100 mg each of methyl and propyl
paraben) on Day 1 and a tablet containing 150 mg of
each paraben on Day 2. Of the 37 subjects, five exhibited
‘‘immediate reactions’’ to paraben ingestion.
In a review of adverse reactions to food and drug
additives, Bush and Taylor (1999) discussed several
methodological flaws in the clinical studies alleging a
role of parabens in chronic urticaria/angioedema. In
some of these studies, placebo controls were not in-
cluded, while in others, the placebos were administered
first, leading to concerns about false positive reactions.
The concerns about false positive reactions are exacer-
bated by the withdrawal of antihistamines from the pa-
tients employed in these clinical trials. By definition,
chronic urticaria is a recurrent condition that could
flare-up at any time. In many patients, antihistamines
are essential to control the symptoms. The withdrawal
of antihistamines in these patients is likely to result in
breakthrough urticaria. Breakthrough urticaria could
result in false positives when the placebos are always
administered before the test materials. The possible role
of ingested parabens in the causation of chronic urti-
caria/angioedema and allergic vascular purpura remains
to be established.
Several cosmetics or topical medications containing
parabens have been implicated in a number of cases of
contact sensitization, usually either dermatitis or urti-
caria (Hjorth and Trolle-Lassen, 1961, 1962; Scham-
berg, 1967; Agrup, 1969; Aeling and Nuss, 1974;
Hannuksela et al., 1976; Marzulli and Maibach, 1976;
Simpson, 1978; Henry et al., 1979; Fisher, 1982; Warin
and Smith, 1982; Simon, 1989; Smith, 1991; Nielsen
and Menne, 1992, 1996; Fisher, 1996; Soni et al., 2001,
2002). The role of parabens in contact dermatitis and
contact urticaria is rather well substantiated in many
of these reports through the use of patch testing to doc-
ument localized sensitization after prolonged exposure
to one or more of the parabens. However, the patch tests
were often conducted at high concentrations of the para-
bens (5–15%). The relevance of the study results is ques-
tionable as the usual concentration of the parabens in
cosmetics is 0.1–0.3% and in foods is usually limited to
a maximum of 0.1%. The use of parabens in foods raises
few concerns regarding contact sensitivity, because the
ingested parabens are unlikely to stay in contact with
mucosal surfaces for a sufficient period to cause adverse
reactions, as parabens are rapidly metabolized.
Judd et al. (1982) reported three cases of autoanti-
bodies with Jka specificity using a single manufacturerÕs
low-ionic-strength salt (LISS) solution containing
methyl and propyl paraben as preservatives. Tests with
other commercially prepared LISS reagents (both dilu-
ent and additive types) were non-reactive, as were con-
ventional saline-albumin and enzyme tests. In a more
recent study, Judd et al. (2001) reported propyl paraben
was capable of manifesting the paraben-dependent
antiRh in a 24-year-old woman. Reports of autoanti-
bodies with Jka (Kidd A) in individuals with Jk (a+)
blood cells are rare.
2.3.5. Paraben paradox
Several issues related to paraben usage and allergen-
icity has been referred to as the ‘‘paraben paradox’’
(Fisher, 1996). One such issue is that individuals who
show positive test results to parabens on patch testing
and develop dermatitis on compromised skin, can freely
use paraben containing products on other areas of body
that have intact, uninvolved skin. This may be the result
of sensitization from products that have higher concen-
trations of paraben compared with lower concentrations
of paraben in cosmetic products. This paradox may be
from the use of topical medicaments on compromised
skin, allowing for easy penetration of allergen, vs. the
use of paraben-containing cosmetic products on intact
skin, which allows relatively less penetration. The sec-
ond paradox is that paraben sensitive individuals can
tolerate injected or oral medication that contains para-
bens. The third paradox is that in burn patients, applica-
tion of paraben containing medication does not result in
a higher incidence of sensitization. The possible explana-
tion for the later paradox may lie with the role that Lan-
gerhans cells play in eliciting acute contact dermatitis. In
burn patients, Langerhans cells are rapidly destroyed,
hence injected or oral medications bypass the Langer-
hans cells.
 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
The chemical structure of parabens suggests that they
are generally incapable of producing sensitization and
induction of immediate or delayed hypersensitivity with-
out prior conjugation to a carrier molecule, usually a
protein. The bound paraben with protein is then consid-
ered a hapten. Although the capacity of simple chemi-
cals to form in vivo covalent bonds with the carrier
molecule correlate with their ability to induce antibody
formation and delayed hypersensitivity, this chemical
property does not necessarily correlate with its ability
to produce immediate hypersensitivity or anaphylactic
reactions. Presence of a free amino group at the para po-
sition of the benzene ring of chemicals such as p-amino-
benzoic acid, p-phenylenediamine, benzocaine and
procaine makes them frequent sensitizers. Structurally,
parabens differ from these chemicals by having a hydro-
xyl group rather than an amino group in the para posi-
tion. Cross sensitivity between p-amino and p-hydroxyl
substituted compounds has been reported.
2.3.6. Estrogenic activity in humans
In recent years there has been a growing concern
about the exposure of humans to synthetic estrogens.
Some investigators claim that the functional abnormal-
ities of the immune system, neurological defects, or
appearance of tumors that are noticed in adult life
may be linked to exposure to toxic agents, including
estrogens, in the pre-natal period. The cause of de-
creased sperm count in males has been suggested to be
exposure to elevated concentrations of estrogens or
estrogen-like substances during embryonic, fetal and
early post-natal development. It has been hypothesized
that synthetic chemicals with estrogenic activity are eti-
ologic factors in abnormalities of the male reproductive
system and an increased incidence of breast cancer in
women.
As discussed earlier, in vivo animal (oral and subcu-
taneous) and in vitro studies indicate that parabens
may mimic estrogen-like activity. Recently, Darbre
et al. (2004) reported minimal detection (in nanogram
quantities) of parabens in a small number (n = 20) of
samples of human breast tumor tissue. As parabens
may possess estrogen like activity, these investigators
indicated that their presence in breast tissue may con-
tribute to the rising incidence of breast cancer. This
and other studies raise concern of possible endocrine
disrupting potentials of parabens. These findings and
its potential implications for humans as to (1) develop-
mental/reproductive changes and (2) breast cancer are
discussed below.
2.3.6.1. Developmental and reproductive effects. In vitro
and in vivo animal studies are described earlier (see Sec-
tion 2.2.9). However, in order to understand its implica-
tions for humans, these studies are briefly discussed
here. In a series of in vitro studies with yeast-based
1007
estrogen assay, estrogen binding receptor assay and
estrogen receptor-dependent proliferation of human cell
lines, parabens showed weak estrogenic activity. In an
in vivo uterotrophic assay, subcutaneous administration
of butyl paraben showed weak estrogenic effects in
immature rats. Interestingly, oral administration of
butyl paraben did not show a uterotrophic response.
Similarly, subcutaneous administration of p-hydroxy-
benzoic acid, primary metabolite of parabens, to mice
produced vaginal cornification and increased uterine
weights. In sexually immature rainbow trout parabens
(methyl, ethyl, propyl and butyl) showed estrogenic
activity, while p-hydroxybenzoic acid was negative. In
one study, subcutaneous administration of parabens
did not produce uterotrophic effects in immature mice,
while at high doses a weak estrogenic response was
noted in rats.
Subcutaneous administration of butyl paraben to rats
during gestation and lactation adversely affected the F1
male offspring. In oral exposure studies in rodents, Oishi
(2001, 2002a,b) reported adverse effects of propyl para-
ben and butyl paraben. These studies indicate that expo-
sure of newborn male rodents to butyl and propyl
paraben adversely affects the secretion of testosterone
and the function of male reproductive system (decrease
in sperm count). In spite of these reports, no studies
assessing reproductive function were identified. The
available data on reproductive toxicity of parabens is
insufficient to fully characterize potential human health
hazards.
2.3.6.2. Parabens and breast cancer. As estrogen is a ma-
jor etiological factor in the growth and development of
the majority of cases of human breast cancer, it has been
proposed that the use of parabens in cosmetics, particu-
larly underarm deodorants and antiperspirants, may
contribute to the rising incidence of breast cancer. Clin-
ical observations show a disproportionately high inci-
dence of breast cancer in the upper outer quadrant of
the breast, just the local area to which these cosmetics
are applied. Recent results of the presence of paraben
in tumor tissue (Darbre et al., 2004) along with results
of the earlier described studies on the estrogenic poten-
tial of parabens lead to the hypothesis that parabens
may be involved or contribute to the incidence of breast
cancer. In a series of recent studies and reports, this
hypothesis has been extensively discussed (Harvey and
Darbre, 2004; Harvey, 2003; Harvey and Everett,
2004; Darbre et al., 2004).
In a recent study, parabens were detected in a small
number (n = 20) of tissue samples from human breast
tumors (Darbre et al., 2004). Of the parabens detected,
methyl paraben represented approximately 60% of the
total parabens. Other parabens detected were ethyl-,
n-propyl-, n-butyl- and isobutyl-paraben. The mean
concentration of parabens in breast tumors was found
1008 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
to be 20.6 ± 4.2 ng/g tissue (blank value of 33.8 ng/g
subtracted); the corrected (for 50% recovery) average
total concentration of paraben was calculated as
100 ng/g. Methyl paraben was present at the highest
level (with a mean value of 12.8 ± 2.2 ng/g tissue). The
corrected average value was compared with the level
of approximately 150 ng/ml (10 6 M) of propyl-, butyl-
and isopropyl-paraben that have stimulated estrogen-
dependent growth in MCF7 human breast cancer cells.
The argument that levels of parabens could exert estro-
genic effects on cells in human breast may not hold, as
methyl paraben was found at the highest level, which
is much less potent estrogenically. The average level of
three more active parabens (propyl, butyl and isopropyl)
could be estimated as 15 ng/g (corrected for 50% recov-
ery), which is comparable to a concentration of 10 7 M,
but this concentration is not found active in MCF7
assay. Although results from Darbre et al. (2004) study
are only preliminary, the clinical significance cannot be
ascertained. In this study, no control tissues were exam-
ined to demonstrate specificity of association with breast
cancer. These studies need to be corroborated as
‘‘blanks’’ in this study had variable levels of parabens,
the study lack in use of control tissues, lack of patient
history of drug administration (which may contain para-
bens), details of breast tumor type, etc.
There have been no published epidemiological studies
to support or refute the hypothesis for a link between
use of underarm perspirant containing synthetic estro-
gens such as parabens and increased incidence of breast
cancer. In a population-based case control study, Mirick
et al. (2002) investigated a possible relationship between
the use of antiperspirants/deodorants and the risk for
breast cancer in women aged 20–74 years. Subjects in
the test group (n = 813) and control group (n = 793)
were studied for use of antiperspirants and deodorants,
and whether they applied them within 1 h of underarm
shaving. Results showed that women who used antiper-
spirants/deodorants and women who shaved their
underarms did not have a greater risk in developing
breast cancer. These initial findings do not support the
hypothesis that antiperspirant use increases the risk of
breast cancer. In another study, McGrath (2003) re-
ported that frequency and earlier onset of usage of
deodorant and underarm shaving were associated with
an earlier age of breast cancer in a survey of 437 females
diagnosed with breast cancer. However, no epidemio-
logical studies specific to parabens and breast cancer
development have been performed.
3. Risk evaluation
The parabens are esters of p-hydroxybenzoate, pre-
pared by esterification of p-hydroxybenzoate with the
corresponding alcohol in the presence of a catalyst. Gen-
erally, parabens are soluble in oil and poorly soluble in
water. As the ester chain length of paraben increases the
water solubility decreases. Parabens are stable in air and
resist hydrolysis in acidic medium and under conditions
of sterilization. Parabens are used as antimicrobial pre-
servatives in foods, drugs and cosmetics for over
50 years. The antimicrobial activity of parabens in-
creases with increasing ester chain length. Parabens, par-
ticularly methyl and propyl paraben, have been
approved for use in food over the past three decades
by the FDA, several foreign governments, the European
Community and JECFA.
Studies in rats, mice, rabbits, dogs and cats show that
parabens are absorbed from the gastrointestinal tract
and metabolized. Parabens are hydrolyzed to p-hydr-
oxybenzoic acid, conjugated and its conjugates are ex-
creted in the urine. The excretion of parabens appears
to be rapid and they do not accumulate in the body.
Most of an administered dose of parabens can be recov-
ered within 5–72 h as p-hydroxybenzoic acid or its con-
jugate. No evidence of accumulation of parabens or its
metabolites was found in acute or chronic studies. Para-
bens appears to be rapidly absorbed through the intact
skin.
Acute toxicity studies in several animal species indi-
cate that parabens are practically non-toxic by various
routes of administration. Parabens, at a concentration
of 10%, were at most mildly irritating when applied to
rabbit skin but were non-irritating at lower concentra-
tions. Subchronic and chronic oral toxicity testing stud-
ies also indicate that parabens are practically non-toxic.
In a chronic study of isobutyl paraben in mice the only
effect was amyloidosis in 58% of dosed males and 33% of
dosed females surviving 78 weeks, as compared to 25%
of control males and 10% females. The majority of the
animal sensitization tests indicate that parabens are
non-sensitizing.
Mutagenicity testing of parabens in the Ames test,
cytogenetic assays, dominant lethal assay and host-med-
iated assay indicate that parabens are not genotoxic.
Methyl paraben was non-carcinogenic by subcutaneous
route in mice and rats. In transplacental carcinogenesis
assays, propyl paraben was non-carcinogenic. In a study
on the urinary bladder tumor-promoting potentials of
propyl paraben, no increased incidence of papillary or
nodular hyperplasia, papilloma and carcinoma was
noted. In a carcinogenicity study of isobutyl paraben,
no changes in either neoplasm incidence or time to neo-
plasm development were observed in mice fed isobutyl
paraben for 102 weeks. The chemical structure of
paraben is not indicative of carcinogenic potential and
parabens are rapidly metabolized and excreted from
the body without accumulation of the parent compound
or metabolites. Developmental toxicity studies in several
species, using methyl paraben, showed no teratological
effects.
 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
In the population with normal skin, parabens are
practically non-irritating and non-sensitizing. However,
application of medications containing parabens to dam-
aged or broken skin has resulted in sensitization. It has
been recognized that preservatives must be used to en-
sure product stability and safety, and that the parabens
are innocuous preservatives (Orth, 1980). This makes
parabens the preservative of choice for most formula-
tions. The widespread use of parabens in cosmetic prod-
ucts made in the United States, without a significant
number of complaints from consumers, gives support
to their safety and effectiveness. It is recognized that
inclusion of methyl paraben and propyl paraben in top-
ical preparations intended for use on compromised skin
is contraindicated. However, use of methyl paraben or
propyl paraben as preservatives in cosmetic products in-
tended for use on normal skin is considered safe.
In 1974, the Joint FAO/WHO Expert Committee on
Food Additives recommended that the group Acceptable
Daily Intake (ADI) for the methyl-, ethyl- and propyl-
paraben was 0–10 mg/kg body weight/day (JECFA,
1974). The basis of the JECFA recommendation of this
value was the consideration of rat feeding study at 2%
in the diet (equivalent to 1000 mg/kg body weight/day).
At this level, no toxicological effects were observed. At
the next highest dose, 8% (approximately 5500 mg/kg)
methyl or propyl paraben in diet only a transient decrease
in weight gain was noted without any toxic or histopatho-
logic findings. The no observed adverse effect level
(NOAEL) for this study may be considered as 5500 mg/
kg. As the effects of any intermediate levels were not avail-
able, JECFA recommended the ADI of 10 mg/day based
on a no observed effect level (NOEL) of 1000 mg/day.
Recent studies suggest that parabens possess estro-
genic potential. As regards the estrogenic potential of
parabens, two important issues need to be addressed:
(1) its reproductive effects and (2) its relation to in-
creased incidence of breast cancer. Few reports and par-
ticularly those from Oishi (2001, 2002a,b) suggest
adverse reproductive effects (such as decrease in epidid-
ymides weight, serum testosterone concentration, sperm
count) of propyl and butyl parabens by the dietary
route. More recent investigations by this author revealed
that methyl and ethyl paraben had no adverse reproduc-
tive effects. Although these studies are from one labora-
tory and the same author, the circumstantial evidence
(particularly the potential estrogenic activity of para-
bens) indicate a possibility of reproductive effects. How-
ever, these studies need to be corroborated. The ability
of parabens to transactivate the estrogen receptor in
vitro increases with alkyl group size. Methyl and butyl
paraben, administered orally to immature rats in an
uterotrophic assay, were inactive. The estrogenic po-
tency of paraben and its metabolite in the uterotrophic
assay by the subcutaneous route in mice and in in vitro
studies ranges from 500- to 10,000-fold less than estra-
1009
diol, respectively. In addition to estrogenic activity,
butyl and isobutyl paraben increased progesterone
receptor gene expression. The estrogenic hazard of para-
bens, on the basis of the available studies is, at best,
equivocal, as it may relate to humans, as metabolism
and elimination rates of parabens are believed to be
dose, route and species dependent.
Based on a recent study, the presence of minute
amounts of parabens in human breast tumor tissues
has been associated to the development of breast cancer.
This study implied a link for use of parabens in under-
arm antiperspirants and deodorants, rather than their
use as food additives. These findings need to be inter-
preted with great care, as trace amounts of widely used
substances can be detected in human tissues (several per-
sistent chemicals with higher estrogenic activity have
been found in human breast tissue without any link to
cancer); secondly, the study indicating the link had sev-
eral limitations (high background, low recovery, absence
of appropriate control) and; thirdly, no association be-
tween breast cancer and use of underarm antiperspirant
and deodorant has been made. However, the potential
estrogenic activity of parabens and detection of para-
bens in breast tissue, albeit from a very small sample,
is noteworthy. However, rather than investigating these
issues (i.e., reproductive toxicity as well as breast can-
cer), as unrelated events, a more comprehensive investi-
gation may be warranted.
Because of the incomplete data on endocrine dis-
ruptive potentials and reproductive toxicity of para-
bens, Harvey and Darbre (2004) proposed a tiered
weight of evidence approach for risk assessment of
parabens. The objective of such an assessment is to
determine if the chemical is clearly unacceptable,
clearly acceptable, or if more data are required. In this
evaluation, the authors proposed use of structure
activity relationships, in vitro estrogen assay, short-
term in vivo uterotrophic assays and available repro-
ductive toxicity studies. However, the limitation for
such an assessment is the lack of reliable reproductive
or developmental toxicity studies, as the existing stud-
ies evaluating these parameters do not meet the cur-
rent standard accepted guidelines. The absence of
systematic toxicity studies meeting the current regula-
tory guidelines can never be taken unequivocally to be
‘‘evidence for the absence of harm’’, it is, nevertheless,
important that this larger perspective be maintained in
assessing the overall safety of paraben uses. A com-
prehensive investigation of the reproductive/develop-
mental toxicity of parabens may be warranted to
assure their continued safe use as preservatives in
foods, cosmetic and toiletries.
Several tests among the standard toxicology studies
are capable of detecting estrogenic activity. Although
reproduction studies should show the effect of hormone
imbalance, multigenerational studies would be the most
1010 M.G. Soni et al. / Food and Chemical Toxicology 43 (2005) 985–1015
sensitive. Avian reproduction studies would also detect
estrogenic activity. In addition, weights of organs with
hormone-sensitive tissue are measured in the range find-
ing, subchronic and chronic studies. Considering that
maximum tolerated doses are used in the toxicology stud-
ies, estrogenic activity relevant to human, animal or eco-
logical effects would be detected in these standard tests.
In summary, parabens are the success story of preser-
vatives, however some recent studies raise concern about
their use. The assessments of a possible reproductive
hazard and suggestion of potential involvement of para-
bens in breast cancer on the basis of the available
published studies, are equivocal. A comprehensive
reproductive toxicity study, meeting the currently ac-
cepted standard protocols for such studies, may be war-
ranted to fully explore possible (if any) human health
risks of parabens.
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