NeuroToxicology 58 (2017) 194–202

Contents lists available at ScienceDirect

NeuroToxicology

Full Length Article

A two-generation inhalation reproductive toxicity study upon the

exposure to manganese chloride
Doreen McGougha,* , Lynne Jardineb
a
International Manganese Institute, France
b
Charles River, United Kingdom

A R T I C L E I N F O A B S T R A C T

Article history:
Received 26 February 2016 A number of published studies have suggested that high levels of exposure to manganese, especially
Received in revised form 22 September 2016 those found in occupational settings, can adversely affect the reproductive system. The objective of this
Accepted 23 September 2016 study was therefore to investigate if these findings can be replicated using the Sprague Dawley rat and, if
Available online 23 September 2016 so, to identify those parts of the reproductive system are more susceptible.
Male and female rats were exposed to manganese dichloride (MnCl2) via inhalation at concentrations
Keywords: of 0 (air-control); 5, 10 and 20 mg/L air over 10 weeks (F0) and over 11 weeks (F1) prior to mating, and
Manganese then throughout mating, gestation and lactation until termination after the F1 and F2 generation had
Reproduction
reached Day 21 of lactation respectively.
Fertility
Animals were monitored for clinical signs of toxicity and for effects on body weight, food consumption,
Rats
MnCl2 effects on the entire reproductive system including maternal care. The offspring were monitored for
Inhalation survival and growth up to weaning. Blood samples were taken from all adult animals for bioanalytical of
manganese analysis prior to dosing, prior to mating and prior to weaning/necropsy.
There were no deaths related to treatment, though respiratory tract effects were observed in F0 animals
in the mid and high dose animals. Body weight and food consumption were affected at high dose in both
generation. There were no treatment-related effects on the oestrous cycles, mating performance, sexual
maturity, fertility or duration of gestation or litter size, the sperm motility, count of morphology (sperm)
or the ovary follicle scoring in either generation.
The No Observed Effect Level (NOEL) for reproductive performance was considered to be the target
dose level of 20 mg/L.
Based on these findings, manganese chloride could not be considered a reprotoxicant under these
conditions of exposure. Therefore, soluble and insoluble forms of inorganic manganese compounds by
extrapolation cannot be considered as reprotoxicants.
ã 2016 Elsevier B.V. All rights reserved.

1. Introduction administration of radiolabelled MnCl2 to pregnant maternal

Manganese (Mn) is a naturally occurring and abundant element
that is essential in biological systems but deficiency or excess of
this mineral can lead to adverse effects. From a toxicity
perspective, toxicity driven by excess has primarily been reported
by exposure via the inhalation route, mainly in workplace studies
(Ellingsen et al., 2003a,b; Wirth and Mijal, 2010; Egorova, 2009).
Several publications have indicated that manganese ions cross the
placenta reaching embryos, as the manganese content of
embryonic tissues was elevated following intravenous
* Corresponding author.
E-mail address: doreen.mcgough@manganese.org (D. McGough).
animals (Koshida et al., 1965; Onoda et al., 1978). Placental transfer
was also demonstrated after inhalation exposure of maternal
animals (Dorman et al., 2000). Inhalation of MnSO4 in pregnant
rats resulted in a clear elevation in manganese levels in the
maternal animal, but only a small increase in livers of the embryos
(Dorman et al., 2005). Although MnCl2 and MnSO4 are very soluble
compounds, both exhibiting a valency of +2, marked differences in
reaction to treatment have been seen when different manganese
compounds were administered: while studies with MnCl2
demonstrated effects on fertility and juvenile development
(Elbetieha et al., 2001; Treinen et al., 1995) no effects were
observed with MnSO4 administered at considerably higher doses
in some studies, a noteworthy study is a 2-year study in rats and
mice, indicated no change in testes weight or in the seminiferous

http://dx.doi.org/10.1016/j.neuro.2016.09.017
0161-813X/ã 2016 Elsevier B.V. All rights reserved.
 D. McGough, L. Jardine / NeuroToxicology 58 (2017) 194–202
tubules upon the exposure to manganese sulphate at concen-
trations up to 15,000 ppm (NTP, 1993a,b).
There are reports that young animals may take up more
manganese than adults, but the situation is not completely clear
and may just reflect a need of the neonate for more manganese to
synthesize enzymes (Fechter, 1999). The importance of considering
the essentiality of manganese on reproduction has been stressed
by several authors as manganese deficiency in humans’ results in
significant adverse consequences including growth impairment,
reduced fertility and risk of birth defects, i.e. some of the very
effects that have been suggested to associate with manganese
excess.
Overall, the quality of published studies on the effects of
manganese on reproductive toxicity varies. Most of these studies
are neither good quality nor conducted in compliance with any
established regulatory guidelines; with several confounders in
some cases. Some report reproductive effects mainly in doses that
cause maternal toxicity without making the link and others show
effects with exposure via intravenous, subcutaneous or intraperi-
toneal routes, which are not relevant means of exposure in the
workplace or to the general population. This is reflected in the
current assessments by authoritative bodies (SCOEL, 2013; ATSDR,
2012) which concluded that, the evidence on the effects of
manganese on reproductive functions is conflicting and inconclu-
sive.
Therefore, a well-designed animal study by the most relevant
route of exposure in the workplace with adequate doses
(specifically a guideline compliant inhalation two generation
reproduction study) was necessary to draw a robust conclusion.
MnCl2 was chosen as the test substance as it is both soluble and the
most toxic as demonstrated by several experimental rodent
studies.
2. Materials and methods
2.1. Introduction
This study is designed to fulfil the requirements of OECD
Guideline 416 and US EPA Guideline OPPTS 870.3800 while
investigating any possible reproductive effects upon exposure to
manganese especially in the workplace where oral and dermal
exposure are negligible.
Manganese dichloride (MnCl2) was selected as an appropriate
test material for this study because it is very water soluble and seen
to produce greater general toxicity compared to MnSO4 according
to available data as discussed in the introduction.
Doses were selected after a nose-only inhalation exposure dose
range finder study. Ten females and 10 males per group were
exposure to manganese chloride at 5, 20 and 30 mg/L. The study
was scheduled for 9 weeks however; exposure was stopped at 3
weeks for the high dose as adverse clinical signs included
Table 1
Experimental design.
Dose Group/ Target Concentration (mg/L Air)— F0 Actual Concentration (mg/L Air)— F1 Actual Concentration (mg/L Air)— Number of Animals
Treatment Nose only Nose only
1a Air Control 0 0
2 Low Dose 5 6
3 Intermediate 10 15
Dose
4 High Dose 20 25
a
For Group 1, Manganese (II) Chloride was non-detectable or non quantifiable for all samples collected over the course of the study.
195
crackling/gasping respiration resulted in the premature sacrifice
of 3 animals. Necropsy findings included distended intestines,
froth filled trachea, discoloured lungs. Exposure continued to the
end of the study for the low and mid dose animals with minimal
effects. The high dose for the main study was therefore reduced to
20 mg/L.
In the main study, F0 animals were randomised into 3 test
groups and one control group, each containing 28 males and 28
females (see Table 1). These animals were dosed for 10 weeks (6 h
daily) prior to mating, and then throughout mating, gestation and
lactation until termination after the F1 generation had reached Day
21 of lactation.
From each treatment group, at least 24 males and 24 females
were retained for post weaning assessments. These animals
continued on the study (F1) and were dosed for approximately
11 weeks (6 h daily) after weaning, and throughout mating,
gestation and lactation until termination after the F2 generation
had reached Day 21 of lactation.
Animals were monitored for clinical signs of toxicity and for
effects on body weight, food consumption, effects on oestrous
cycles, mating performance, pregnancy performance, difficulty or
prolongation of parturition, and for deficiencies in maternal care.
The offspring were monitored for survival and growth up to
weaning. In addition, the following endpoints were evaluated:
gross necropsy findings, organ weights, histopathology evaluation,
qualitative examination of testes and examination of the ovaries
and sperms.
2.2. Animal husbandry
One hundred and fourteen male and 114 female Sprague-
Dawley rats (Crl:CD1(SD)) were housed in cages. Cages were
racked by treatment group with males and females racked
separately. The F0 animals were allowed to acclimate for 13 days
before the commencement of dosing.
For at least 7 days prior to the commencement of dosing, all
animals were conditioned to the restraint procedures (to adapt to
the inhalation chamber) used for nose-only exposure by placing
the animals in the restraint tubes for gradually increasing periods
of restraint time up to the maximum expected duration to be used
on the study—6 h daily.
SDS Rat and Mouse (modified) No. 3 Diet SQC Expanded and tap
water was provided ad libitum to test and control animals
throughout the study, except during designated procedures. Each
batch of diet was routinely analysed by the supplier (supplied by
SDS Special Diets Services) for various nutritional components and
chemical and microbiological contaminants, including manganese,
which is a known essential element in the SDS Rat and Mouse diet
and the levels of manganese were (84–94 mg/kg) in each batch of
diet. The drinking water was periodically analysed for dissolved
materials, heavy metals, pesticide residues, pH, nitrates, nitrites
Nose only
F0 F0 F1 F1
Males Females Males Females
0 28 28 26 26
4 28 28 24 24
10 28 28 24 24
17 28 28 25 25
196 D. McGough, L. Jardine / NeuroToxicology 58 (2017) 194–202
and selected bacteria. Results of these analyses did not provide
evidence of contamination; however, Mn is a known component of
potable water and was present at a concentration of 1.9 mgMn/L.
Therefore additional Mn exposure from diet and water intake is
expected.
2.3. Test material preparation
The test material MnCl2 was purchased from Sigma Aldrich
Limited in the form of a 99.0% pure pink powder. It was passed
through a centrifugal grinder using the finest mesh available and
then sieved using a mesh the size of 100 or 180 mm prior to use.
Test item aerosols were generated using a Wright Dust Feed
generator device (Wright Dust Feed Mark II, BGI Industries, USA).
2.4. Administration of test materials
F0 animals commenced treatment with MnCl2 at 6–8 weeks of
age, and were treated for 10 weeks prior to pairing for mating.
Daily exposure to MnCl2—nose only, was for 6 h, 7 days a week.
Meanwhile F1 animals commenced treatment shortly after
weaning for 11 weeks (prior to mating). Daily exposure was for
6 h, 7 days a week using a modular nose only stainless steel flow
past inhalation chamber.
For F0 and F1 males, this treatment continued until the day
prior to termination (a total of 17 weeks per generation).
For F0 and F1 females, the animals were dosed throughout
gestation up to and including Day 19 of gestation. The animals were
not dosed from Day 20/21 of gestation until their litters were born
and then exposure was initially reduced to allow the dams to
acclimatise to being away from their litter. The females were then
dosed as follows:
From Day 1–2 of lactation: for 1 h per day
From Day 3–4 of lactation: for 2 h per day
From Days 5–20 of lactation until prior to termination (ca Day
21 of lactation): for 6 h per day.
Animals that did not produce litters, continued on a 6 h dosing
until the scheduled termination. Animals that had a litter loss
continued on a 6 h dosing regimen until scheduled sacrifice.
A few days prior to the initiation of mating, the males were
separated into individual grid bottomed cages. Pairings were on a 1
male to 1 female basis. Animals were paired in numerical order
within the groups. Each female was transferred to the cage of its
appropriate co-group male near the end of the work day, where it
remained until mating had occurred or 14 days had elapsed.
Vaginal lavages were taken daily early each morning from the day
of pairing until mating occurred and the stage of oestrous observed
in each lavage recorded. The presence of sperm in such a lavage
and/or a copulatory plug in situ was designated as Day 0 of
gestation.
The females were allowed to litter normally. If any animal
suffered from a difficult or prolonged parturition, this was
recorded. The day of birth of the litter (day on which the first
pups are born) was designated Day 0 of lactation. The duration of
gestation was calculated.
The numbers of live and dead pups born in each litter was
recorded as soon as possible after completion of parturition on Day
0 of lactation. The live pups were counted and examined from Day
1 onwards for the presence of milk in the stomach and for any
externally visible abnormalities daily. The pups were weighed en
masse, sexes separated, on Days 1, 4, 7 and 14 of lactation. On Day
21 all pups were weighed individually.
Where practicable, any pups that were found dead or were
killed during lactation were sexed and examined as above. Prior to
Day 14 of lactation, any externally abnormal decedent pup was
preserved; externally normal ones were discarded. On or after Day
14 of lactation, decedent pups were necropsied.
2.5. Observations: animals were monitored for clinical signs of toxicity
and of effects
Body weight, food consumption, effects on the entire repro-
ductive system including maternal care. Organs below were
collected and preserved in 10% neutral buffered formalin, unless
otherwise indicated in Table 2:
The tissues assigned to histological examination were proc-
essed; sections were cut 4, stained with haematoxylin and eosin
(H&E) and evaluated by light microscopy from animals in the
Controls and High dose groups:
Additionally, a Periodic Acid Schiff and Haematoxylin (PAS-H)
stained section was prepared from the left testis.
A detailed qualitative examination of the testes was made,
taking into account the tubular stages of the spermatogenic cycle.
Computer assisted sperm analysis was also conducted to assess the
motility of the sperm. Sperm counts using a haemocytometer to
obtain a total sperm count was assessed. A sperm smear was
prepared and stained with eosin and evaluated for morphological
abnormalities. One testis was decapsulated and homogenized and
the homogenisation resistant spermatids were counted using a
haemocytometer.
The examination of the ovaries included quantification of the
primordial and growing oocytes, and the confirmation of the
presence or absence of the corpora lutea.
Histological examination was conducted on the brain, spleen
and thymus of Control and High dose F1 and F2 weanlings (the
selected weanlings at necropsy). A single Haematoxylin and Eosin
(H&E) section was cut, stained and evaluated
2.6. Blood analysis
Blood samples were taken from the tail vein of all adult animals
for analysis of Mn concentrations prior to dosing, prior to mating
and prior to weaning/necropsy. Samples were collected from all F0
generation animals at pre-dose, prior to mating and prior to
weaning/necropsy. Meanwhile for the F1 generation, samples were
collected from all animals (timing and volume depended on weight
of animals, prior to mating and at necropsy. All samples were
stored at 80  C and analyzed for Mn in whole blood via ICP-MS.
2.7. Statistical analysis
Where required to assist with interpretation, tests were applied
to determine the statistical significance of observed differences
Table 2
Organs collected and preserved for observations of clinical signs of toxicity.
Animal identification Lymph node, bronchialc
Brainb Lymph node, cervicalc
Epididymis x 2b,c Nasal cavityc
Gland, adrenal x 2b,c Ovary x 2b,c
Gland, pituitaryb Pharynxc
Gland, prostateb,c Spleenb
Gland, seminal vesiclec Testesa,b,c
Gland, thyroid x 2b Trachea (anterior)c
Kidney x 2b Trachea (posterior)c
Larynxc Uterusb,c
Liverb Vaginac
Lungb,c
a
Preserved in Modified Davidson’s fixative.
b
Weighed.
c
Evaluated by light microscopy in Control and High dose animals.
 D. McGough, L. Jardine / NeuroToxicology 58 (2017) 194–202 197

between Control and groups receiving test item. Unless otherwise
stated, all statistical tests were two-sided and performed at the 5%
significance level using in house (validated) software. Pairwise
comparisons were only performed against the control group.
Select body weight and food consumption were analysed for
homogeneity of variance using the ‘F-Max’ test. If the group
variance appeared homogeneous, a parametric ANOVA was used
and pairwise comparisons were made using Fisher’s F-protected
LSD method via Student’s t-test ie pairwise comparisons was made
only if the overall F-test was significant. If the variances were
heterogeneous, log or square root transformations were used in an
attempt to stabilize the variances. If the variances remained
heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was
used and pairwise comparisons were made using chi squared
protection (Via z tests, the non-parametric equivalent of Student’s t
test).
Organ weight data was analysed as above, and by analysis
of covariance (ANCOVA) using terminal body weight as the
covariate.
3. Results
3.1. Inhalation results
The overall aerosol concentrations aerosols were 6, 15 and
25 mg/L for the F0 Generation and 4, 10 and 17 mg/L for the F1
Generation. This was considered to be satisfactorily close to target
concentrations of 5, 10 and 20 mg/L for Groups 2, 3 and 4,
respectively.
For the air-alone control group (Group 1), MnCl2 was non-
detectable or non-quantifiable for all samples collected over the
course of the study.
The particle size distribution (mass median aerodynamic
diameter (MMAD) and geometric standard deviation (GSD))
investigations assured that the test aerosols were respirable to
the animals and that good pulmonary exposure was achieved. See
Table 3 below:
Particle size distribution investigations assured that the test
aerosols were respirable to the animals and that good pulmonary
exposure was achieved.
Table 3
Group mean particle size distribution measurements for F0 and F1.
Dose Group/Treatment Cumulative% <3.4 mm MMAD (GSD) (mm)
3.2. Mortality
Four animals in the F0 generation (one Control, 2 at target 5 mg/
L and one at 10 mg/L) and 5 animals in the F1 generation were (one
Control, 3 at target 10 mg/L and 1 at target 20 mg/L) killed
prematurely for various reasons –difficulties giving birth, reduced
activity, partially closed eyes, unkempt coat, open lesion on tail etc.
There was no treatment-related pattern to these deaths and these
were not attributed to treatment.
3.3. Clinical observations
3.3.1. F0 animals
One female animal in Group 3 had clinical signs including
wheezing, unkempt coat, walking on tip toes, rolling gait and
weight loss recorded over Days 83–90 of the study. Due to the signs
dosing for the animal was stopped for a two days. However, the
animal recovered from these signs and dosing continued until
scheduled termination. As no similar findings were noted in the
other animals in this group or in group 4, these signs were
considered to be incidental. Blood concentrations correlated with
exposure levels in males and females (Tables 4 and 5) confirming
incremental systemic test material availability.
At target 20 mg/L (group 4) there were 2/28 males noted as
having wheezing respiration.
Other clinical signs noted in the F0 animals were considered to
be incidental or due to the dosing procedure (wet, unkempt coat).
3.3.2. F1 animals
Clinical observations noted in the F1 animals were considered
to be incidental or due to the dosing procedure (wet, unkempt
coat).
3.4. Body weight
3.4.1. F0 animals
At target dose 20 mg/L, there was a decrease in body weight gain
in males over Days 0–21 of the study (29% lower gains than those of
the Controls). From Day 21 of the study, the body weight gains
were generally comparable to the controls but the group mean
weights remained lower than the controls throughout the study.
At target dose 20 mg/L, the group mean body weight gain in
females, prior to mating were similar to the controls, however
body weight gains over Days 0–20 of gestation were slightly lower
than the controls (9% of the Controls). Gains over the lactation
period were similar to the controls.

Gravimetric Analytical Gravimetric Analytical 3.4.2. F1 animals

2 Low Dose 50.9 53.8 2.13  2.502 2.32  2.567
At target dose 20 mg/L, there was a reduction in group mean
3 Intermediate Dose 48.1 53.7 2.51  2.383 2.44  2.465 body weight gain of the males during the first 5 days of the study
4 High Dose 49.3 55.2 2.48  2.194 2.57  2.398 (p < 0.001), however gains over the following week were greater
MMAD = Mass Median Aerodynamic Diameter. GSD = Geometric Standard Devia- than the controls and then remained comparable with the controls
tion. throughout the remainder of the treatment period.

Table 4
Blood Manganese Concentration, F0 and F1 Males.

Time-point Blood Manganese Concentration (ppb w/v (ng/mL)

Group 1a (Control) Group 2 (5 mg/L) Group 3 (10 mg/L) Group 4 (20 mg/L)
F0 Pre-treatment 7 7 7 6
F1 Pre-treatment 12 16 16 17
F0 Prior to mating 6 13 23 27
F1 Prior to mating 6 9 13 19
F0 Prior to Necropsy 6 19 27 29
F1 Prior to Necropsy 6 9 14 21
a
Note: Control animals were exposed to manganese via their food and water.
198 D. McGough, L. Jardine / NeuroToxicology 58 (2017) 194–202

Table 5
Blood Manganese Concentration, F0 and F1 Females.

Time-point Blood Manganese Concentration (ppb w/v (ng/mL)

Group 1a (Control) Group 2 (5 mg/L) Group 3 (10 mg/L) Group 4 (20 mg/L)
F0 Pre-treatment 7 7 7 7
F1 Pre-treatment 13 12 15 15
F0 Prior to mating 6 16 28 39
F1 Prior to mating 6 10 16 23
F0 Prior to Necropsy 7 16 24 33
F1 Prior to Necropsy 7 10 16 21
a
Note: Control animals were exposed to manganese via their food and water.

Slight intergroup differences in group mean body weight gains 3.6.4. Ovary scoring
in the F1 females prior to mating were too small to be attributed to There was no treatment-related effect on the follicle type or
treatment. At 20 mg/L, there was a slight reduction in body weight incidence in either generation. The scoring methodology included
gains throughout gestation compared to the controls. the quantification of the primordial and growing oocytes, and the
There were no effects of treatment noted in the lactating confirmation of the presence or absence of the corpora lutea.
females.
3.7. Litter size and pup mortality
3.5. Food consumption
3.7.1. F0 generation, F1 production
3.5.1. F0 animals
The mean number of implant sites and total number of pups
At target dose 20 mg/L, there was reduced food consumption for
born in all groups was comparable to controls.
males throughout the majority of the study, compared with the
At target 20 mg/L, there was an increase in the number of
controls.
animals losing more than 2 pups at birth (total pups born/no. of
At target dose 20 mg/L, there was a transient reduction in food
implantation sites). However, the mean birth index (%) was well
consumption in the females on commencement of treatment
within the historical background range, hence these increases
compared with the controls; however, consumption for the
were considered to be incidental.
remainder of the pre-mating period was similar to the controls.
Slight intergroup differences in the group mean food consump-
3.7.2. F1 generation, F2 production
tion in the males at target dose 5 mg/L and target 10 mg/L were not
The mean number of implant sites and total number of pups
attributed to treatment.
born in all groups was comparable to controls.
Slight intergroup differences in group mean food consumption
At target 10 and 20 mg/L, pup survival (no. losing >3 pups) over
throughout gestation and lactation were not attributed to
Days 0–4 of lactation was slightly lower than the controls.
treatment.
However, the number of animals losing the entire litter was
comparable with controls and the remaining animals generally lost
3.5.2. F1 animals
4 pups. In addition, there was no clear dose-related response to
At target dose 20 mg/L, there was a slight reduction in group
these reductions and these were considered not to be an effect of
mean food consumption in the males over Days 40–68 of the
treatment.
study; these reductions achieved statistical significance (ranges
from p < 0.05 to p < 0.001).
Slight intergroup differences in group mean food consumption 3.8. Litter and pup weights
at target 5 mg/L and target 10 mg/L were no attributed to treatment.
Group mean food consumption in the females prior to mating 3.8.1. F0 generation
and throughout gestation and lactation were comparable to the In all treated groups, group mean litter and pup weights were
controls. comparable to the controls.

3.6. Reproductive parameters 3.8.2. F1 generation

At target 20 mg/L, group mean litter weights were slightly lower
3.6.1. Oestrous cycles than the controls; this was considered a reflection of the smaller
The stages of the oestrus cycles and their mean duration were litter size and not an adverse effect. This interpretation was
similar in all Test groups compared to the control group for both supported by the mean pup weights in both males and females
generations. being comparable to the controls.

3.6.2. Mating performance, fertility and duration of gestation 3.8.3. Abnormalities among pups
There were no treatment-related effects on mating perfor- The type and distribution of observations amongst pups did not
mance, fertility or duration of gestation in either F0 or F1 indicate any association with treatment if compared to species
generation. historic data.

3.6.3. Sperm assessment 3.8.4. Sexual maturation

There were no treatment-related effects on the sperm motility, The age and body weight at preputial separation or vaginal
count of morphology at any of the dose levels applied, or in either opening of the F1 generation animals in all treated groups was
generation. similar to the controls.
 D. McGough, L. Jardine / NeuroToxicology 58 (2017) 194–202
3.9. Organ weight
3.9.1. Adult organ weights
3.9.1.1. F0 animals
3.9.1.1.1. Brain. At target 20 mg/L, reduced absolute brain weights
in males achieved statistical significance (P < 0.05) compared with
controls. However, the lower body weight was also statistically
significant (P < 0.05) and following covariance analysis; brain
weight did not achieve significance and therefore was not
positively attributed to treatment.
3.9.1.1.2. Lung. In all treated females, there was a statistically
significant increase in absolute lung weights, compared with the
controls; these increases were still present following covariance
analysis with body weight (P < 0.01 at target 5 mg/L and P < 0.001
at target 10 and 20 mg/L).
3.9.1.1.3. Other. Other slight differences in organ weights such as
an increased thyroid weight in males at target dose of 5 mg/L (only)
and an increase in kidney weights of females at target 10 mg/L were
not attributed to treatment. These effects were not seen in the high
dose groups of either sex. The absence of dose-dependent effects
means that these results could not be considered to treatment
related.
3.9.1.2. F1 animals
3.9.1.2.1. Kidneys. At target doses 5 and 10 mg/L, kidney weights in
males were statistically higher than the control (p < 0.01 and
p < 0.05, respectively), however there was no dose-response
relationship to this increase and following covariance analysis
with body weight, these findings were no longer evident.
At target doses 10 and 20 mg/L, there was a statistically
significant increase in kidney weights in females (P < 0.05 at target
10 mg/L and P < 0.001 at target 20 mg/L) following covariance
analysis with body weight.
3.9.2. Weanling organ weights
3.9.2.1. F0 generation, F1 production. At target 20 mg/L, there was a
reduction in thymus weight of the females, compared with the
controls (P < 0.01). Following covariance analysis with body
weight, this reduction did not achieve statistical significance.
There were no effects on any organ weights at target doses of 5
and 10 mg/L.
3.9.2.2. F1 generation, F2 production. Slight intergroup differences
in organ weights did not achieve statistical significance and were
not attributed to treatment.
3.10. Necropsy findings
3.10.1. F0 and F1 adults and F1 and F2 weanlings
There were no treatment related gross findings recorded. The
findings observed were considered incidental, of the nature
commonly observed in this strain and age of rat, and/or were of
similar incidence in control and treated animals and, therefore,
were considered unrelated to administration of MnCl2.
3.11. Histology
3.11.1. F0 and F1 adults
There were no treatment related findings observed in the
reproductive tract in the F0 or F1 generations.
Histological findings related to treatment were confined to the
respiratory tract as follows:
199
3.11.2. F0 animals
3.11.2.1. Larynx. In the larynx there was a broadly dose-related
minimal to moderate squamous metaplasia with minimal to
moderate submucosal inflammation. Minimal to marked
ulceration of the laryngeal epithelium was associated with the
squamous metaplasia in several animals from all treated groups.
Occasional incidences of mineralisation, intraluminal necrotic
debris or intra-epithelial pustules were seen in some of the treated
animals.
3.11.2.2. F0 lungs. In the lungs, the principal MnCl2-related change
was seen in centroacinar regions where there was minimal or mild
inflammation and focal or diffuse minimal or mild broncho-
alveolar hyperplasia. This latter finding was considered reactive.
Minimal or mild goblet cell hyperplasia in the bronchial or
bronchiolar epithelium was present in animals exposed to target
20 mg/L together with occasional incidences of degeneration and/
or squamous metaplasia of the bronchiolar epithelium. Minimal
inflammatory findings (inflammatory cell foci and perivascular
inflammatory cell infiltration) were also present with a greater
incidence in animals exposed to MnCl2 than in controls.
3.11.2.3. Nasal cavity. In the nasal cavity, minimal or mild goblet
cell hyperplasia and minimal to moderate eosinophilic globules in
the olfactory epithelium were observed in all the male treated
groups and in females exposed to target 10 or 20 mg/L. At all dose
levels, there was a greater incidence of minimal or mild
submucosal inflammatory cell infiltration compared to controls.
In males, inflammation of the nasolacrimal duct and squamous
metaplasia of the ductal epithelium was seen in most animals
exposed to target 10 or 20 mg/L. In addition to these changes,
incidences of minimal or mild focal degeneration of the olfactory,
respiratory or transitional epithelia, minimal or mild atrophy of the
olfactory epithelium, ulceration and focal squamous metaplasia
were observed, mainly in animals exposed to target 10 or 20 mg/L,
but occasionally in animals at target 5 mg/L. Deposits of crystalline
material, presumed to be test item, was seen in the nasolacrimal
ducts of a few animals in the treated groups.
3.11.2.4. Pharynx. Minimal goblet cell hyperplasia was observed in
the pharynx of most males exposed to target 20 mg/L and there
were occasional incidences of minimal or mild focal epithelial
degeneration, focal inflammation and focal squamous metaplasia
in males exposed to MnCl2 at 10 or 20 mg/L.
3.11.2.5. Trachea. In the trachea, minimal or mild focal squamous
metaplasia and inflammation at the carina and minimal or mild
focal epithelial degeneration at sites other than the carina were
observed at all dose levels.
Other microscopic findings observed were considered inciden-
tal, or of the nature commonly observed in this strain and age of rat,
and/or were of similar incidence and severity in control and
treated animals and, therefore, were considered unrelated to the
inhalation of MnCl2.
3.11.3. F1 animals
3.11.3.1. Nasal cavity. Crystals were occasionally observed in the
nasal cavity and in the pharynx from animals exposed to target 10
or 20 mg/L. They consisted of small amounts of needle-shaped
crystals either deposited on the olfactory epithelium in the nasal
cavity, or free in the lumen of the pharynx. These crystals were
considered to result from deposition of MnCl2 in some parts of the
respiratory tract.
200 D. McGough, L. Jardine / NeuroToxicology 58 (2017) 194–202
Squamous metaplasia in the nasal cavity was observed mainly
in the nasolacrimal duct and to a lesser extent in the transitional
and respiratory epithelia.
3.11.3.2. Trachea. In the trachea, findings such as epithelial
degeneration, squamous metaplasia and submucosal
inflammation were observed predominantly in the carina.
Other microscopic findings observed were considered inciden-
tal, of the nature commonly observed in this strain and age of rat,
and/or were of similar incidence and severity in control and
treated animals and, therefore, were considered unrelated to
administration of MnCl2.
3.12. F1 and F2 weanlings
There were no treatment related findings observed in the
tissues examined of the F1 or F2 weanlings.
3.13. Blood analysis
Pre treatment manganese levels in all F0 animals were similar
to controls.
The manganese concentrations in the blood of all the treated F1
animals were lower than the same time-point levels of the F0
generation animals.
The F1 generation pre-treatment Mn blood levels were higher
than the control with dose dependent correlation indicating
exposure prior to treatment via lactation. Milk Mn levels were not
measured directly but Mn blood pre-treatment levels confirm Mn
exposure prior to weaning.
4. Discussion and conclusions
Clinical signs of reaction to treatment to inhalation exposure of
MnCl2 were confined to a few animals with wheezing respiration
in the F0 generation exposed to target levels of 10 and 20 mg/L. This
could be attributed to slower lung clearance rate of the test
material and or the presence of free chloride ions aggressive to the
lung lining causing local reactions and even lesions.
At target 20 mg/L, overall body weights and food consumption
of the F0 males throughout the study were lower than controls;
this is not unusual for long term exposure studies if dosing is high
enough. In fact such effects at highest dose are important markers
for dose appropriateness.
In the F1 generation, the body weight gain of the males at target
20 mg/L were transiently reduced on commencement of treat-
ment; in addition, the food consumption at this level was lower
than the controls over Days 19–68 of treatment. Meanwhile in
females, at target 20 mg/L, there was a slight reduction in group
mean body weight gains during gestation in both generations.
Gains throughout lactation were similar to controls. No con-
clusions could be drawn from this finding with regards to sex
dependent susceptibility as the gains in males was transient and in
females, increase or decrease in weight gain during gestation or
lactation is not an usual phenomena even in humans.
In females, there were no effects of treatment on the sexual
maturity of the F1 animals. There was no effect of treatment on
oestrous cycles, mating performance, ovary follicle scoring,
fertility or duration of gestation or litter size in either generation.
While these findings were very clear and occurred in both
generations – increasing the confidence in the results, some
researchers reported differently at doses 10 times higher that pre-
pubertal exposure to manganese induces precocious puberty in
rats as it induces early elevations in puberty-related hormones
(Dearth et al., 2014) and induces abnormal development of the
reproductive tissues based on puberty related hormonal changes
(Kim et al., 2012). Others have reported no effects on hormonal
changes such as prolactin (Ellingsen et al., 2007). These differences
in findings could be associated to the extrapolations that hormonal
changes have a direct correlation on the growth and development
on specific reproductive tissues and that they are not expected to
adjust over time. However, this study did not investigate
reproductive hormones per se but looked at actual effect on
reproductive parameters at different stages of development at set
doses, in which it concludes no effects on female reproductive
parameters in both generations.
With regards to group mean litter and pup weights in the F0
generation, both were comparable with controls. This is an
important investigative parameter as several factors (from food
consumption to hormonal changes and general toxicological
effects of F0 animals) can affect litter and pup weight. Therefore
the absence of comparable difference between exposed animals
and the control is an affirmation of no significant reproductive
effect at F0 generation upon the exposure to the test material.
However, at target 20 mg/L, group F1 mean litter weights were
slightly lower than the controls, but this was attributed to the
slightly smaller litter sizes. The mean pup weights in both males
and females were comparable to the controls hence the slightly
lower litter weights were not attributed to treatment.
In males, there were no effects of treatment on the sexual
maturity of the F1 animals. There was no effect of treatment on the
sperm motility and count of morphology (sperm) in either
generation. Many studies investigating male fertility upon the
exposure to manganese in the workplace (Li et al., 2012a,b; Meeker
et al., 2010; Pizent et al., 2012; Wirth and Mijal, 2010) and in
animals (Chandra et al., 1973; Seth et al., 1973; Gennart et al., 1992;
Lauwerys et al., 1985; Laskey et al., 1982) have reported
contradictory findings. While many of the animal studies did
not report with enough scientific detail or used a routes of
exposure not relevant for occupational settings or general
population exposure route, such as intra-tracheal or intravenous
administration, from the well reported studies, most of the male
fertility findings occurred at doses above those reported to cause
neurotoxicity, hence these findings have been considered to be
secondary. Studies on occupational settings have also reported
contradictory findings on male fertility either due to the presence
of several confounders such as other metallic compounds in the
environment or just smaller samples sizes and differences in
methodology and interpretation of results. Hence the conclusions
from this well designed animal study on the relevant route of
exposure should be considered as a departure point for evaluating
any possible effect on the reproductive parameters upon the
exposure of manganese with the highest dose of 20 mg/L
considered as the No Observed Effect Level (NOEL).
At organ level, the target doses 10 and 20 mg/L, produced
statistically significant increase in kidney weights compared to the
controls. The structure of the Kidneys was not affected at 10 mg/L
but an alteration was seen microscopically (at target 20 mg/L).
However, this was not considered a treatment related effect as
structural specific changes were random and it was not statistically
significant.
Microscopic changes were seen in the respiratory tract – which
was not surprising as this was the port of entry. Effects were seen in
the nasal cavity, larynx, lung and trachea (including carina), and
pharynx in the F0 and F1 generations. It was thus concluded that,
apart from the lungs, there were no systemic toxic effects due to
MnCl2 up to 20 mg/L.
No MnCl2-related findings were observed in the reproductive
tract in the F0 or F1 generations and in tissues examined from
weanlings in the F1 and F2 generations. While this is contradictory
to some findings in published literature showing effects especially
in the male reproductive system, many of such cross-sectional
 D. McGough, L. Jardine / NeuroToxicology 58 (2017) 194–202
epidemiological studies are faced with confounders such as
alcohol intake. Mergler et al. (1994) reports that manganese
may have greater effects on heavy drinkers. The liver health status
of the subjects is another important confounder in some of these
studies. A report from WHO (1981) commented that serum
manganese levels were increased in the active phase of hepatitis.
Also, Montes et al. (2001) reports that rats with liver disease show
increased accumulation of manganese in the brain and dopamine
metabolism – factors that can trigger neurotoxic effects leading to
reproductive effects as a secondary outcome. Analysing for
anaemia is another parameter which some of these studies fail
to examine – Mena et al. (1974) concludes that anaemic subjects
may take up twice as much manganese as those who are not
anaemic. From an environmental exposure stand point the
presence of other metallic compounds and life style are parameters
of concern but not present in this study. This study, in a control
environment is not faced with such confounding factors hence
there was no need for adjustments in the finding making the result
a reliable point of exposure departure below which reproductive
effects are not expected.
In all treated groups of the F0 generation, the levels of MnCl2 in
the blood increased significantly on commencement of dosing in
both males and females. The concentrations recorded prior to
mating and prior to necropsy were comparable in all groups which
did not indicate any obvious accumulation over the dosing period.
This is probably the reason researchers reported blood not to be a
good biomarker of exposure as it is quickly eliminated from the
body. In the F1 generation, pre-treatment concentrations in all
groups were higher than the F0 generation pre-treatment values.
In addition, at target 5 and 10 mg/L in the F1 generation, the pre-
treatment values were generally higher or similar to the values
recorded during the dosing period, indicating that the exposure to
MnCl2 through the mother’s milk during lactation resulted in an
increased exposure to the test item in the F1 animals from birth. At
target 20 mg/L, the Mn concentrations of the F1 males and females
throughout the dosing period were greater than the pre-treatment
values indicating an increased exposure throughout the dosing
period.
To summarise, respiratory tract effects are commonly observed
in irritant materials especially if exposure is via inhalation. This
was therefore, not considered to be a unique toxicological effect
from MnCl2 but more an effect from the slower lung clearance of
the Mn2+and the irritating effect from the Cl2. The parental No
Observed Adverse Effect Level (NOAEL) was considered to be target
level 20 mg/L and the No Observed Effect Level (NOEL) for
reproductive performance was considered to be at target dose
level 20 mg/L.
This study adds to the several reports on the effects of
Manganese on the reproductive system in humans with some
reports suggesting that when men are exposed to Manganese for a
longer period of time they may show signs of loss of libido and
impotence and impaired fertility (ATSDR, 2012). The ATSDR report
which analysis a large body of published literature also suggests
that impaired sexual function in men may be one of the earliest
clinical manifestations of manganese toxicity. This study has
provided a confident threshold for the adverse effects on male and
female reproductive performance and capacity that toxicity is not
expected to arise.
It is clear from this study that below the 20 mg/L exposure level,
no reproductive effects would be seen. This value can be
extrapolated to determine a safety exposure limit for inorganic
manganese substances with regards to reproduction. Interpreting
results from reproductive toxicity studies can be daunting as
general maternal toxicity, genetic susceptibility, life style, effects
on both sexes are all important parameters to be considered but
they can vary considerably.
201
Conflict of interest
None.
Acknowledgement
We will like to thank the International Manganese Institute,
Paris- France and its members for sponsoring this research.
References
ATSDR, 2012. Agency for Toxic Substances and Disease Registry Toxicological Profile
for Manganese. ATSDR Available at: http://www.atsdr.cdc.gov/toxprofiles/
tp151.pdf.
Chandra, S.V., Ara, R., Nagar, N., Seth, P.K., 1973. Sterility in experimental manganese
toxicity. Acta Biol. Med. Ger. 30 (6), 857–862.
Dearth, R.K., Hiney, J.K., Srivastava, V.K., et al., 2014. Prepubertal exposure to
elevated manganese results in estradiol regulated mammary gland ductal
differentiation and hyperplasia in female rats. Exp. Biol. Med. (Maywood) 239
(7), 871–882.
Dorman, D.C., Struve, M.F., Vitarella, D., Byerly, F.L., Goetz, J., Miller, R., 2000.
Neurotoxicity of manganese chloride in neonatal and adult CD Rats following
subchronic (21-day) high-dose oral exposure. J. Appl. Toxicol. 20, 179–187.
Dorman, D.C., McElveen, A.M., Marshall, M.W., Parkinson, C.U., Arden James, R.,
Struve, M.F., Wong, B.A., 2005. Maternal-fetal distribution of manganese in the
rat following inhalation exposure to manganese sulfate. Neurotoxicology 23,
625–632.
Egorova, A.M., 2009. A reproductive function in metallurgists. Urologiia 65–68.
Elbetieha, A., Bataineh, H., Darmani, H., Al-Hamood, M.H., 2001. Effects of long term
exposure to manganese chloride for fertility of male and female mice. Toxicol.
Lett. 119, 193–201.
Ellingsen, D.G., Haung, E., Ulvik, R.J., Thomassen, Y., 2003a. Iron status in manganese
alloy production workers. J. Appl. Toxicol. 23, 239–247.
Ellingsen, D.G., Hetland, S.M., Thomassen, Y., 2003b. Manganese air exposure
assessment and biological monitoring in the manganese alloy production
industry. J. Environ. Monit. 5, 84–90.
Ellingsen, D.G., Chashchin, V., Haug, E., Chashchin, M., Tkarenko, V., Lubnina, N.,
Bast-Pettersen, R., Thomassen, Y., 2007. An epidemiological study of
reproductive function biomarkers in male welders. Biomarkers 12 (5), 497–509.
Fechter, L.D., 1999. Distribution of manganese in development. Neurotoxicology 20,
197–201.
Gennart, J.P., Buchet, J.P., Roels, H., Ghyselen, P., Ceulemans, E., Lauwerys, R., 1992.
Fertility of male workers exposed to cadmium, lead or manganese. Am. J.
Epidemiol. 135 (11), 1208–1219.
Kim, S.I., Jang, Y.S., Han, S.H., et al., 2012. Effect of manganese exposure on the
reproductive organs in immature female rats. Dev. Reprod. 16 (4), 295–300.
Koshida, Y., Kato, M., Hara, T., 1965. Distribution of radiomanganese in embryonic
tissues of the mouse. Annot. Zool. Jpn. 38 (1), 1–7.
Laskey, J.W., Rehnberg, G.L., Hein, R.F., Carter, S.D., 1982. Effects of chronic
manganese (Mn3O4) exposure on selected reproductive parameters in rats. J.
Toxicol. Environ. Health 9, 677–687.
Lauwerys, R., Roels, H., Genet, P., Toussaint, G., Bouckaert, A., De Cooman, S., 1985.
Fertility of male workers exposed to mercury vapor or to manganese dust: a
questionnaire study. Am. J. Ind. Med. 7, 171–176.
Li, P., Zhong, Y., Jiang, X., Wang, C., Zuo, Z., Sha, A., 2012a. Seminal plasma metals
concentration with respect to semen quality. Biol. Trace Elem. Res. 148 (1), 1–6.
Li, Y., Wu, J., Zhou, W., Gao, E., 2012b. Effects of manganese on routine semen quality
parameters: results from a population-based study in China. BMC Publ. Health
12, 919.
Meeker, J.D., Rossano, M.G., Protas, B., 2010. Environmental exposure to metals and
male reproductive hormones: circulating testosterone is inversely associated
with blood molybdenum. Fertil. Steril. 93 (1), 130–140.
Mena, I., Horiuchi, K., Lopez, G., 1974. Factors enhancing entrance of manganese into
the brain: iron deficiency and age. J. Nucl. Med. 15, 516.
Mergler, D., Huel, G., Bowler, R., Iregren, A., Bélanger, S., Baldwin, M., Tardif, R.,
Smargiassi, A., Martin, L., 1994. Nervous system dysfunction among workers
with long-term exposure to manganese. Environ. Res. 64, 151–180.
Montes, S., Alcaraz-Zubeldia, M., Muriel, P., Ríos, C., 2001. Striatal manganese
accumulation induces changes in dopamine metabolism in the cirrhotic rat.
Brain Res. 891, 123–129.
NTP Technical Report, 1993a. Toxicology and carcinogenesis studies of manganese
(II) Sulphate mononhydrate (CAS No. 1003-96-5) in F344/N rats and B6C3F1
Mince, NTP TR 428 NIH Publication. No. 94-3159 U.S. Department of Health and
Human Services, pp. 5–272.
NTP Technical Report, 1993b. Toxicology and carcinogenesis studies of manganese
(II) Sulphate monohydrate (CAS No. 1003-96-5) in F344/N rats and B6C3F1
Mince, (feed studies), NIH Publication. No. 94-3159 NTP TR 428 U.S. Department
of Health and Human Services.
Onoda, K., Hasegawa, A., Sunouchi, M., TanakaA. Omori, Y., Urakubo, G., 1978. studies
on the fate of poisonous metals (VII)—distribution and transplacental passage of
manganese in pregnant rat and fetus. J. Food Hyg. Soc. 19 (2), 208–2015.
Pizent, A., Tariba, B., Zivkovic, T., 2012. Reproductive toxicity of metals in men. Arhiv
za Higijenu Rada i Toksikologiju 63 (Suppl.1), 35–46.
202 D. McGough, L. Jardine / NeuroToxicology 58 (2017) 194–202

EC Employment, Social Affairs and Inclusion, 2013. Methodology for the Derivation
of Occupational Exposure Limits. Scientific Committee on Occupational
Exposure Limits (SCOEL) Key Documentation version 7.
Seth, P.K., Nagar, N., Hussain, R., Chandra, S.V., 1973. Effects on manganese on rabbit
testes. Environ. Physiol. Biochem. 3, 263–267.
Treinen, K.A., Gray, T.J., Blazak, W.F., 1995. Developmental toxicity of mangafodipir
trisodium and manganese chloride in Sprague-Dawley rats. Teratology 52 (2),
109–115.
WHO, 1981. Manganese (International Programme on Chemical Safety,
Environmental Health Criteria, 17). World Health Organization, Geneva,
Switzerland.
Wirth, J.J., Mijal, R.S., 2010. Adverse effects of low level heavy metal exposure on
male reproductive function. Syst. Biol. Reprod. Med. 56 (2), 147–167.