Accepted Manuscript

Evaluation of embryotoxic and teratogenic effects of the oil extracted from Caryocar
brasiliense Cambess pulp in rats

Giseli Karenina Traesel, Fernando Freitas de Lima, Ariany Carvalho dos Santos,
Roosevelt Isaias Carvalho Souza, Daniela Torres Cantadori, Carlos Roberto
Kretschmer, Vinicius João Navarini, Silvia Aparecida Oesterreich
PII: S0278-6915(17)30614-2
DOI: 10.1016/j.fct.2017.10.018
Reference: FCT 9343

To appear in: Food and Chemical Toxicology

Received Date: 16 August 2017

Revised Date: 9 October 2017
Accepted Date: 11 October 2017

Please cite this article as: Traesel, G.K., de Lima, F.F., dos Santos, A.C., Souza, R.I.C., Cantadori,
D.T., Kretschmer, C.R., Navarini, Vinicius.Joã., Oesterreich, S.A., Evaluation of embryotoxic and
teratogenic effects of the oil extracted from Caryocar brasiliense Cambess pulp in rats, Food and
Chemical Toxicology (2017), doi: 10.1016/j.fct.2017.10.018.

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Evaluation of embryotoxic and teratogenic effects of the oil extracted

from Caryocar brasiliense Cambess pulp in rats

Giseli Karenina Traesela*, Fernando Freitas de Limaa, Ariany Carvalho dos Santosa,

Roosevelt Isaias Carvalho Souzaa, Daniela Torres Cantadoria, Carlos Roberto

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Kretschmerb, Vinicius João Navarinic, Silvia Aparecida Oesterreicha

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a
Faculty of Health Sciences, Federal University of Grande Dourados, Dourados, Mato

Grosso do Sul, Brazil

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b
Course of Veterinary Medicine, Faculty Anhanguera Dourados, Dourados, Mato

Grosso do Sul, Brazil

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Universitary Hospital, Federal University of Grande Dourados, Dourados, Mato Grosso
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do Sul, Brazil
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*Corresponding author. Faculty of Health Sciences, Federal University of Grande

Dourados. Rodovia Dourados - Itahum, Km 12. Caixa Postal - 533. CEP: 79.804-970.

Dourados, MS, Brazil. Phone: + 55 67 34102332. E-mail address:

giselitraesel@gmail.com
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Abstract

The objective of this study was to evaluate the maternal, embryotoxic and teratogenic

effects of Caryocar brasiliense pulp oil (OPCB), oil widely used in Brazilian cuisine

and traditional medicine. Pregnant Wistar female rats were used in this study for three

treatment groups (250, 500 and 1000 mg/kg/day) and a control group. The OPCB was

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administered orally throughout the period of organogenesis of females (6th until the 15th

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day of gestation). The pregnant females were gross necropsied on d20, followed by

maternal and fetus examination, to evaluate the teratogenicity, reproductive and

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developmental performance of OPCB. The results showed there was no significant

statistical difference in the ponderal evolution of the pregnant females, as well as in the

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behavioral, hematological, biochemical or histopathological data, indicating the absence
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of maternal toxicity of the oil. The mean number of corpora lutea, implantation and
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resorption sites, as well as all calculated reproductive rates, also remained statistically

similar between the groups, indicating low embryotoxic effects of the tested plant
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specie. In fetal examination, external anomalies and skeletal abnormalities were

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observed in all treated and control groups. The NOAEL for maternal toxicity and

embryo/fetal development for the OPCB administered by gavage, was 1000

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mg/kg/bw/day.
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Keywords: pequi; safety; pregnancy; preclinical studies; Brazilian folk medicine; food

toxicology.
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Abbreviations:

AAP: adequate for age of pregnancy; ALT: alanine aminotransferase; ANOVA:

analysis of variance; AST: aspartate aminotransferase; FID: flame ionization detector;

LAP: large for age of pregnancy; LD50: oral lethal dose; MCH: mean corpuscular

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hemoglobin; MCHC: mean corpuscular hemoglobin concentration; MCV: mean

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corpuscular volume; NOAEL: no-observed-adverse-effect-level; OPCB: oil from the

pulp of Caryocar brasiliense; RDW: red cell distribution width; SAP: small for age of

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pregnancy; SEM: standard error of the mean; UFGD: Federal University of Grande

Dourados.

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Highlights:
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* The toxicological potential of Caryocar brasiliense pulp oil was investigated through

preclinical studies.
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* The Caryocar brasiliense pulp oil was administered orally throughout the period of
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organogenesis.

* No maternal, embryotoxic and teratogenic effects was observed for Caryocar

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brasiliense pulp oil.

* The NOAEL of Caryocar brasiliense pulp oil were 1g/kg bw/day, the highest dose
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tested.
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1. INTRODUCTION

The use of plant-based products in primary health care continues to expand and

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gain worldwide popularity. This use is justified by the therapeutic benefits derived from

the active ingredients present in plant species (Ekor, 2013). Although these plant

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products are popularly considered safe, in fact, it is known that the active components

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can cause serious adverse effects, mainly because they do not undergo rigorous safety

tests, as with allopathic drugs (Barnes, 2003). The risk of adverse effects from

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medicinal plants is greater in people belonging to certain at-risk groups, such as the
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elderly, children, fetuses and pregnant women (Essiet et al., 2017).

After the thalidomide tragedy (McBride et al., 1962), developmental toxicity

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tests began to gain visibility in the scientific community. It is now known that
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alterations caused in the embryofetal period can lead to several future developments,
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such as loss of gestation, malformations, functional alterations or neurobehavioural

disorders. Several agents are related to teratology: infectious agents, chemical agents,
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physical agents and also plant species (Lima, 2014). There are numerous reports of

medicinal plants that are known to impair fetal development when consumed by the
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pregnant woman (Nath et al., 1992; Mabina et al., 1997; Rodrigues et al., 2011; Costa et
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al., 2012).

Caryocar brasiliense, Cambess, is a species native to Brazil, popularly known as

"pequi" or "piqui". It is a fruit and oleaginous species, belonging to the family

Caryocaraceae, whose tree can reach 15 meters in height. Its geographic dispersion

includes the entire Brazilian territory, being of wide incidence in the “Cerrado” region
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(Lima et al., 2007). Pequi pulp is used for extraction of high quality oil, used as

functional food and medicinal plant. Pequi oil is a substance widely used in the context

of natural medicine, with reports of effects of balm on rheumatism (Pozo, 1997), anti-

inflammatory, healing effects on the treatment of respiratory diseases, gastric ulcers,

muscular and rheumatic pains (Bezerra et al. Al., 2015). Studies of pharmacological

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effects indicate that the plant performs antigenotoxic, anticlastogenic, chemopreventive,

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anti-inflammatory and hypocholesterolemic activities (Traesel et al., 2016).

Several toxicological studies have been conducted for this species. The toxicity

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of aqueous extracts of fruit peels (65.5 at 500 mg/kg/bw) and leaves (18.75 at 300

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mg/kg/bw) were evaluated in males and females mice (Mus musculus) Swiss by
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intraperitoneal route. The higher dose administered in both experiments were lethal for

all animals. The LD50 corresponded to 149.8 mg/kg/bw for fruit peel extract and 67.01
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mg/kg/bw for the leaf extract, both being classified as very toxic (Fonseca et al., 2016).

Regarding studies on fruits, safety data vary according to the extraction method. Castro
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et al. (2008) examined the genotoxic effects of aqueous extracts of pequi pulp on wing
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spots of Drosophila melanogaster using the test SMART. Seventy-two-hour larvae

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from two crosses were treated with pequi pulp extract at 1, 5 and 10%. The extract

increased significantly the frequency of mutant spots when compared with the negative
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control. Recombinogenic effects were also observed in the descendants. Data show that
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aqueous extract of pequi is genotoxic to Drosophila melanogaster and that mitotic

recombination proved to be the major event responsible for this genotoxicity in the

SMART assay (Castro et al., 2008).

In relation to the oil, data point to absence of genotoxicity. Comet assay and

micronucleus test were performed in blood and bone marrow of Wistar rats treated

orally with a 125, 250, 500, or 1000 mg/kg/bw of the oil from the pulp of C. brasiliense
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for 4 weeks. The tests performed showed that oil did not show significant genotoxic or

clastogenic effects in cells analyzed with the four doses tested (Traesel et al., 2017).

Also in relation to fruit oil, LD50 was established above 2000 mg/kg and there are

reports of no toxicity following subchronic treatment in Wistar rats with doses of 125 -

1000 mg/kg/bw (Traesel et al., 2016). However, to our knowledge, the literature on

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toxicity of pequi development, as an important part of the preclinical toxicology profile,

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is non-existent. Therefore, due to the wide empirical use of pequi oil and lack of

knowledge on the reproductive toxicity of this substance, we investigated the toxicity of

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the embryofetal development and the teratogenic potential of oil extracted from

Caryocar brasiliense pulp in Wistar rats.

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2. MATERIAL AND METHODS
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2.1. Plant collection and identification

C. brasiliense fruits were collected in a Cerrado area from Campo Grande, Mato
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Grosso do Sul-Brazil (latitude 20° 26' 34'' South and longitude 54° 38' 47'' West)
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according to a permit issued by the Brazilian Environmental Agency (Nº 54442-1—

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MMA/ICMBio/SISBIO). The fruits were selected according to their state of maturation.

A voucher specimen was authenticated by Dr.ª Zefa Valdivina Pereira and deposited
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(Nº 4752) in the Herbarium of the Federal University of Grande Dourados (UFGD).
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2.2. Preparation of oil

The fruits were transported to the Laboratory of Food Technology of the Faculty

of Exact Sciences and Technology (UFGD) and were sanitized with water and sodium

dichloroisocyanurate dihydrate 0.66 % (SumavegR) for 5 min. The pequi fruits were

manually depulped and dehydrated in an air circulation oven at 40° C for 72 h. The
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dried pulp was cold-pressed in an ‘‘expeller’’ press to obtain the oil. The oil was

centrifuged at 15.000 g for 15 min for complete separation of sediment. The oil was

then packed in amber glass containers and kept at -8° C for further analysis.

2.3. Chemical analysis of oil

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The oil was previously analyzed by our research group and the results are

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already published. The determination of the fatty acid values was carried gas

chromatograph equipped with autosampler and flame ionization detector (FID) (Traesel

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et al., 2016), as well as characterization of carotenoids through high-performance liquid

chromatography with diode-array detector (Traesel et al., 2017).

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2.4. Animals
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Forty-four Wistar rats (Rattus norvegicus) from both sexes (36 female and 8

male), at the same age (8-10 weeks) and weight (235 g ± 10%) were used in this study.
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The animals were obtained from the UFGD and were housed in polypropylene rodent
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cages with dimensions of 48.3 x 33.7 x 21.4 cm, under standard conditions (23 ± 2° C,

40-60 % humidity and 12 h light and dark cycle) and had free access to water and food.
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The experimental procedures were performed in accordance with the Ethical Principles

in Animal Research and approved by the Ethics Committee in Animal Experimentation

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from the UFGD (protocol: 17/2015).

2.5. Determination of the Estrus Cycle Phase

Vaginal smear analysis test was used to verify the estrus stage of females. A

micropipette containing 10 µL of saline solution was gently and superficially inserted

into the vaginal cavity of females. The saline solution was injected, aspirated and
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immediately transferred to a glass slide and examined fresh under light microscopy. The

proestrus phase was confirmed by the predominance of cornified cells and absence of

leukocytes under microscopic examination (Vilela et al., 2007).

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2.6. Mating and Gestation Confirmation

After confirmation of the estrus cycle phase, females in proestrus were placed

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with proven fertile males in a separate mating cage at the proportion of 2:1 during the

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night time. The next morning, the vaginal smear was redone. The presence of

spermatozoa was taken as an indication of successful mating and that day was

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considered as day 0 of gestation (d0) (Amaral; Nunes Junior, 2008).
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2.7. Experimental Design

Prenatal developmental toxicity study described below was based on the OECD
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protocol (Organisation for Economic Co-operation and Development) – Guideline 414

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(OECD, 2001).

All rats with confirmed pregnancy were individually weighed and divided into
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four groups. The first group (n=6) was designated as the control group and treated with
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saline solution. The other groups (n=7) were treated with 250, 500 or 1000 mg/kg body
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weight of Caryocar brasiliense pulp oil (OPCB). The doses used in this study were

chosen based on previous safety information, such as LD50 above 2 g⁄kg and NOAEL

for subchronic toxicity of 1 g⁄kg⁄bw (Traesel et al., 2016). Saline solution and oil were

orally administered to females throughout the period of organogenesis, which comprises

from d6 to d15 of gestation.

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2.8. Maternal observations

Throughout the treatment period, females were daily observed in relation to

general health and clinical signs of maternal toxicity, in particular attention to vaginal

bleeding. Body weight, water and feed consumption were measured every two days

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(OECD, 2001).

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2.9. Euthanasia and C-section

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On day 20 of gestation (d20), females were sacrificed under inhalation

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anesthesia (isuflurane) followed by exsanguination. The collected blood was used for
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hematological and biochemical analysis. The internal organs of females were removed,

weighed and submitted to histopathological processing in order to detect evidence of

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maternal toxicity.
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After collection of blood and organs, cesarean section was performed with
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hysterectomy and bilateral oophorectomy. Ovaries were weighed and carefully analyzed

for the number of corpora lutea. The gravid uterus was removed whole and weighted.
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All fetuses were individually removed and their placentas were analyzed. The number

of fetuses was counted and several parameters were observed, such as fetal weight,
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placental weight, number of implantations, number of resorptions and number of live

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and dead fetuses. Data were used to calculate reproductive rates: rate of implant

efficiency, pre-implantation loss, post-implantation loss and placental index. The

newborns were classified small (SAP), adequate (AAP) and large (LAP) for age of

pregnancy, according to the body weights (Calderon et al., 1992). All fetuses were

submitted to external morphological analysis and half of each litter was processed for

skeletal analysis.
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2.10. External Morphological Analysis of Fetuses

Under a stereomicroscope, newborns were submitted to systematic analysis for

the detection of possible external structural malformations. Several fetal parameters,

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such as ocular defects, cranial defects, limb defects (including reflex test), abdominal

distension, gastroschisis, hydrops, tail defects and presence of haemorrhages, clots or

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bruises were examined (Wilson, Warkani, 1965; Et al., 2016).

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2.11. Skeletal Analysis of Fetuses
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Immediately after external analysis, half of each litter was placed in 70% alcohol

(12 h), transferred to acetone P.A. (24 h), eviscerated, diaphanized, stained with 1.5%
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aqueous potassium hydroxide solution, added of alizarin red (24 h) and then stored in
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100% glycerol, as previously described (Staples, Schnell, 1964 and Chahoud, 1996).
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The skeleton was systematically analyzed from head to tail. The bones of skull,

vertebrae, ribs, bones of the sternum, forelegs and hind limbs were carefully analyzed.
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Bones were analyzed for bone quantification, number, size, shape, and location.

Ossification centers were also observed and counted (Aliverti et al., 1979).
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2.12. Statistical analysis

The results are reported as mean ± standard error of the mean (SEM) and the P-

values less than 0.05 were set as the level of significance. Statistical analysis of

offspring data was performed using the litter as a unit. The normality of the samples

was checked using the Kolmogorov–Smirnov test. The data with normal distribution
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were evaluated by analysis of variance (one-way ANOVA) followed by Tukey test. The

non-parametric data were analyzed by Kruskal-Wallis, complemented with the Dunn

test. The Qui-Square test was used to compare the percentages of SAP, AAP e LAP.

The statistical analyses were performed by GraphPad Prism software version 5.00 for

Windows.

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3. RESULTS

3.1. Maternal Observations

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All copulated females survived until the end of the study. Initial body weight

(d0), weight gain during oil administration (d6-d15), weight gain throughout the study

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(d0-d20), and total and corrected weight (d20) of all groups treated with OPCB were
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equivalent to data from the control group (Table 1 and Figure 1). During the study
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period, no clinical or behavioral signs indicating systemic, localized toxicity or

miscarriage were observed. Females maintained daily intakes of feed and water in all
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groups throughout the treatment period (Table 1). At the end of the study (d20), all
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females were submitted to euthanasia. Maternal organs were analyzed for absolute and

relative weight and in no evaluation, statistically significant difference was found (Table
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2). After macroscopic analysis, organs were processed and sent to histopathological

analysis, which also did not show any morphological changes (data not shown). With
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blood collected, the hemato-biochemical analysis was performed. Biochemical

parameters remained statistically similar across all experimental groups (Table 3),

similarly to haematological parameters (Table 4).

Table 1. Maternal findings of the female rats treated orally with the oil of Caryocar
brasiliense during the 6th until the 15th day of gestation.

Figure 1. Evaluation of weight gain of the female rats treated orally with the oil of
Caryocar brasiliense during the 6th until the 15th day of gestation. The data are
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expressed as mean ± SEM, n= 6-7 animals/group. The values do not differ from one
another at the level of 5% (ANOVA/Tukey test).

Table 2. Absolute and relative organ weight of the female rats treated orally with the oil
of Caryocar brasiliense during the 6th until the 15th day of gestation.

Table 3. Biochemical parameters in blood of the female rats treated orally with the oil
of Caryocar brasiliense during the 6thuntil the 15th day of gestation.

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Table 4. Hematological parameters in blood of the female rats treated orally with the oil
of Caryocar brasiliense during the 6th until the 15th day of gestation.

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3.2. Uterine and Litter Observations

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Uterine observations and litter parameters of pregnant females are presented in

Table 5. No significant differences were observed in reproductive and embryofetal

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parameters (rate of full term pregnancy, number of corpora lutea, number of
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implantations, rate of implant efficiency, pre and post-implantation loss, resorptions

late, live/dead fetuses, fetal weight, placental weight and placental index) between
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treatment and control groups The sex ratio (M: F) of fetuses examined was 1:0.92

(Control); 1:0.736 (250 mg/kg); 1:1 (500 mg/kg) and 1:1.916 (1000 mg/kg), data being
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within normal reference values (0.00 - 2.81). The classification of newborns in relation
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to body weight also remained statistically similar among groups (Figure 2).
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Table 5. Embryo–fetal toxicity related to reproductive findings of the female rats

treated orally with the oil of Caryocar brasiliense during the 6th until the 15th day of
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gestation.
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Figure 2. Percentage (%) of newborns classified small (SAP), adequate (AAP) and
large (LAP) for age of pregnancy, from female rats treated with the oil of Caryocar
brasiliense during the 6th until the 15th day of gestation. The values do not differ from
one another at the level of 5% (Qui-Square).

3.3. Evaluation of External Abnormalities

During necropsy, careful macroscopic observations were performed in all

fetuses. Three fetuses from the control group presented external alterations (two with
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bruises and one with developmental delay), corresponding to 5.66%. The 250 mg/kg

group had four fetuses with alterations (three with bruises and one with developmental

delay), corresponding to 5.97%. Two fetuses from the 500 mg/kg group presented

alterations (one fetus with bruises and one fetus with distended abdomen),

corresponding to 2.85%. The 1000 mg/kg group had two fetuses with alterations (both

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with bruises), corresponding to 2.85%. Figure 3 illustrates some abnormalities found.

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Figure 3. Evaluation of external abnormalities of the newborns of the female rats
treated orally with the oil of Caryocar brasiliense during the 6th until the 15th day of

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gestation. (A) Newborn of the group 1000 mg/kg presenting hemorrhage. (B) Newborn
of the group control presenting developmental delay (on the right).

3.4. Skeletal Abnormalities

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Skeletal analysis was initiated by counting the ossification centers of all

newborns processed in alizarin red. The mean values found were 29.33 ± 1.307
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(Control), 28.91 ± 2.226 (250 mg/kg), 28.82 ± 2.219 (500 mg/kg) and 31.98 ± 0.466
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(1000 mg/kg). The detailed count of ossification centers is found in Table 6, and all data
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recorded were similar between treatment and control groups. Following the counting of

ossification centers, the detailed analysis of the whole skeleton of fetuses was carried
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out. The findings included several variations (for example: incomplete ossification of

some bones, vertebra in dumbbell, sternebra misaligned or bipartite, ribs wavy or

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supernumerary) and some malformation (change in shape of some bones, absent of

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sternebra or ribs, shortening of forelimbs and hindlimbs). These changes were

statistically similar to the control group and were considered spontaneous and incidental

in nature (see Table 7). Figure 4 illustrates some abnormalities found.

Table 6. Counting of centers of ossification of the newborns of the female rats treated
orally with the oil of Caryocar brasiliense during the 6th until the 15th day of gestation.
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Table 7. Summary of major malformations and minor skeletal variations in the
newborns of the female rats treated orally with the oil of Caryocar brasiliense during
the 6th until the 15th day of gestation.

Figure 4. Evaluation of skeletal abnormalities of the newborns of the female rats treated
orally with the oil of Caryocar brasiliense during the 6th until the 15th day of gestation.
(A) Newborn of the group 250 mg/kg presenting incomplete ossification of bone nasal.
(B) Newborn of the group control presenting misshaped of bone supraoccipital. (C)
Newborn of the group 500 mg/kg presenting absent of one rib. (D) Newborn of the

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group 1000 mg/kg presenting ribs supernumerary bilateral. (E) Newborn of the group
control presenting ribs wavy. (F) Newborn of the group 500 mg/kg presenting two
sternebra bipartite.

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4. DISCUSSION

It is estimated that the prevalence of congenital malformations is about 3 to 5%

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of live births worldwide. The cause of most malformations is still unknown; however,
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chromosomal abnormalities (genetic factors), teratogenic (environmental factors), and

multifactorial inheritance (genetic and environmental factors) are the most important
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identified etiological agents (de Souza Mendes et al., 2015; Harris, 2015). There is an

increasing infant mortality rate in children less than one year of age associated with
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congenital anomalies, which has generated great impact to health services and serious
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repercussions on the life of children and their families. About 30% of pediatric
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hospitalizations are related to health problems due to congenital malformations (de

Souza Mendes et al., 2015).

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The indiscriminate use of medicinal plants during pregnancy is also relevant in

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the context of teratology. Plant products can cause uterine contractile stimuli, trigger

hormone responses or produce genotoxic, mutagenic, fetotoxic and teratogenic effects

(Campesato, 2005).

The aim of the present study was to evaluate the toxicity potential of Caryocar

brasiliense pulp oil development in pregnant Wistar rats and their offspring. Initially,
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before reproductive evaluation, maternal toxicity was evaluated. Many physiological

changes in the pregnant organism can influence the metabolism and excretion of

chemical substances, which can lead to an exacerbated response when compared to the

non-pregnant organism. Insufficient body mass gain during pregnancy can also result in

intrauterine growth restriction or compromise fetal ossification (Lima, 2014).

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Treatment with OPCB in the organogenesis period was not able to cause clinical

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signs of toxicity in the animals tested. Toxicity is manifested through changes in the

weight development of animals, reduction in water or food intake and behavioral

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changes (OECD, 2001). In this study, all parameters observed along and at the end of

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the oil treatment (weight evolution, water and feed consumption, clinical signs, absolute
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/ relative and anatomical / pathological weight of maternal organs, hematological and

biochemical variables) were similar in all groups in relation to negative control. These
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data, together, indicate that pequi oil did not cause any level of maternal toxicity at any

of the doses tested. A study carried out with Momordica charantia L. extract, popularly
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known for its therapeutic properties in the treatment of diabetes, resulted in the
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development of maternal toxicity at doses of 500, 1000 and 2000 mg/kg in Wistar rats
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(Trautenmuller et al., 2017), reinforcing the importance of studies aimed at correlating

the consumption of medicinal plants and maternal toxicological effects.

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The interpretation of changes in reproductive rates may identify the period in

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which reproductive toxic effects have been established (Lourenço et al., 2009). As in the

present study, OPCB administration occurred during the critical period of gestation,

organogenesis, the focus of this work was to analyze changes occurring after the

implantation period. Post-implantation loss refers to the relationship between the

number of implanted blastocysts and those that failed to develop - forming resorptions

and indicating failures in embryonic development (Almeida; Lemonica, 2000). The

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results of this study indicate that the treatment with pequi oil did not affect the number

of resorptions, implantations or live fetuses. Thus, OPCB did not present embryotoxic

or embryoletal effects. However, as already mentioned, the fact that the product is of

vegetal origin does not exempt it from having an embryotoxic effect. Hollembach et al.,

in 2017, for example, investigated the reproductive effects of oregano (Origanum

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vulgare L.) on Wistar rats. The results demonstrated that parental treatment with the oil

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resulted in significant post-implantation losses in treated mothers.

Significant reduction in body mass of newborns is indicative of embryotoxic

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effects (Lima, 2014). In this study, OPCB did not change the intrauterine development

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of fetuses, since fetal weight, placental weight and placental index were not affected.
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The percentage of newborns classified as small, adequate or large for the age of

pregnancy also remained statistically similar across all groups. In addition, fetal growth
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is also positively or negatively influenced by the maternal nutritional status, and in this

study, the progenitors did not have their weight development changed. Thus, data
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obtained corroborate the hypothesis that OPCB administration did not cause any
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embryotoxic effects at any dose tested.

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In the organogenesis period, there is intense cell proliferation and migration,

tissue remodeling and formation of rudimentary organs. This period is characterized as

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the most susceptible to teratogenic agents, with greater probability of occurrence of fetal
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malformations (Brent, 1993). In this study, repeated administration of pequi oil

throughout the organogenesis period did not cause a significant increase in the

frequency of external and skeletal abnormalities in treated animals.

Regarding the external changes observed, both groups (treated and control) had

fetuses with skin bruises. The control group and the 250 mg/kg group had a case of
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developmental delay, while the 500 mg/kg group had a case of distended abdomen. The

observed changes did not exhibit a dose-response relationship and were not significantly

different among groups. These occurrences were therefore considered spontaneous and

incidental.

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For Aliverti et al. (1979), fetal weight is not a conclusive parameter in

developmental studies because it may vary depending on the litter size. For this reason,

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the ossification centers are analyzed, functioning as an additional parameter to evaluate

fetal development. In this study, the counting of ossification centers remained similar in

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both treated and control groups, indicating that fetal growth was not altered after OPCB

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treatment. However, studies with the hydroalcoholic extract of Lantana camara
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demonstrated that this plant was able to increase the frequency of delay in fetal

ossification (Mello et al., 2005), pointing out once again that products of plant origin
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can negatively affect the development of fetuses.

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In the skeletal analysis, the abnormalities found are globally classified as

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variations (change that occurs within the normal population under investigation and is

unlikely to adversely affect survival or health) or as malformations (permanent

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structural change that is likely to adversely affect the survival or health of the species

under investigation) (Chahoud et al., 1999; Solecki et al., 2001). Variations and
C

malformations have been observed in fetuses in all groups and their occurrences were
AC

not dose dependent. Therefore, the observed cases were also considered spontaneous

and incidental.

Many recent studies have been published in order to elucidate the relationship

between consumption of medicinal plants and deleterious effects on the skeletal

composition of fetuses. Honokiol, a major bioactive component isolated from the bark
 18
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of Magnolia officinalis was investigated. No treatment-related external alterations as

well as skeletal malformations were observed in honokiol microemulsion groups

(Zhang et al., 2016). Researchers have verified the effects of tiger milk mushroom

(Lignosus rhinocerotis) on Wistar rats. The skeletal changes found were equivalent

between treated and control groups and were therefore not attributed to treatment (Jhou

PT
et al., 2017). In 2017, researchers found significant skeletal changes attributed to

RI
treatment with oregano (Origanum vulgare L.), indicating the teratogenic effect of this

plant (Hollembach et al., 2017).

SC
In this study, signs of poor ossification and unossified areas were visualized in

U
different bones in animals from OPCB-treated and control groups. Poor ossification is a
AN
skeletal abnormality, classified as a common variation in rodent development (Falk et

al., 2017). The shape change in the supraoccipital bone is a skeletal abnormality
M

classified as malformation (Solecki et al., 2001) and was also visualized in this study.

As this anomaly occurred in control and treatment groups with equivalent incidence, it
D

was not considered as related to treatment. Another alteration observed in this study was
TE

the presence of undulated and/or supernumerary ribs. These findings are classified as
EP

variations and were also visualized in all groups, with no clinical significance.

Finally, the presence of shortening and curvature of the anterior and posterior
C

limbs in different fetuses from the 500 mg/kg group was visualized, being this
AC

abnormality classified as malformation. However, it was observed that all fetuses

affected by this malformation belonged to the same litter. Therefore, it was not possible

to discard the genetic load involved with the presence of this anomaly. In addition,

values were not sufficient to generate statistical significance in relation to the control

group, so that this alteration cannot be associated to treatment. Other malformations and

variations were also investigated and found; however, differences were not statistically
 19
ACCEPTED MANUSCRIPT
significant. Therefore, it is suggested that all skeletal abnormalities found have no

teratogenic significance and are not related to treatment.

In recent years, the importance of oxidative stress as a critical factor in

teratogenesis has been studied. At the beginning of organogenesis, mitochondria are still

PT
immature and the execution of their metabolic function is severely reduced. The

decrease in the antioxidant function results in greater susceptibility of the fetus, leading

RI
to teratogenic events (Hansen and Harris, 2013). In this sense, the importance of the

consumption of vegetal products with chemical composition and antioxidant effects is

SC
highlighted. Pequi pulp oil was previously analyzed by our research group. The

U
analyses indicated a high percentage of oleic acid (56.5% - gas chromatograph equipped
AN
with autosampler and FID) (Traesel et al., 2016), in addition to the presence of β-

carotene and lycopene (10.77 µg/g and 1.63 µg/g respectively, high-performance liquid
M

chromatography with diode-array detector) (Traesel et al., 2017). These data indicate

the high antioxidant capacity of the oil.

D
TE

Some recent studies have been published in order to elucidate the possible toxic

effects of different parts of the Caryocar brasiliense. In vitro cytotoxicity and

EP

phototoxicity of C. brasiliense (supercritical CO2 extract obtained from the leaves)

were assessed using a tetrazolium-based colorimetric assay (XTT) and Neutral Red
C

methods. The results suggest that the extract did not present cytotoxic and phototoxic
AC

hazards (Amaral et al., 2014). Tests performed in Wistar rats (comet assay and

micronucleus test) showed that pequi oil did not show significant genotoxic or

clastogenic effects in cells analyzed with the four doses tested (125, 250, 500 and 1000

mg/kg) (Traesel et al., 2016).

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Based on the absence of maternal toxicity, embryofetotoxicity and

developmental toxicity at any dosage level for Caryocar brasiliense pulp oil

administered by gavage, the highest dose tested: 1000 mg/kg/bw/day was considered as

being NOAEL for maternal and developmental / embryofetal toxicity. Combined with

the reported absence of genotoxicity (Traesel et al., 2017) and general toxicity (Traesel

PT
et al., 2016) of pequi pulp oil, these data provide safety estimates for use during

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pregnancy and provide dosage reference data for further investigations on the safety

profile of this plant.

U SC
5. CONCLUSION AN
Based on the results obtained, treatment with Caryocar brasiliense pulp oil did

not affect maternal weight during any stage of pregnancy, and did not lead to
M

behavioral, hematological, biochemical or histopathological changes during pregnancy.

Consequently, the doses used were not capable of causing maternal toxicity. Treatment
D

also did not affect any rate related to reproductive, embryonic or fetal variables and,
TE

therefore, did not cause embryofetotoxic effect. During pregnancy, it did not prevent
EP

females from having full term pregnancy and did affect the average weight of newborns

or placentas. Treatment also did not interfere with reproductive performance during
C

pregnancy and did not affect the incidence of external and/or skeletal abnormalities.
AC

Treatment with Caryocar brasiliense pulp oil was safe for the mother and offspring of

Wistar rats (Rattus norvegicus), when administered during the period of critical

pregnancy, organogenesis. However, as with thalidomide, which induced severe

teratogenic effects in humans and rabbits, and discrete effects on rodents, further studies

on non-rodent species should be conducted to better define the safety of OPCB use in

humans.
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CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest.

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Table 1
Maternal findings of the female rats treated orally with the oil of Caryocar brasiliense during the 6th until
the 15th day of gestation.

Caryocar brasiliense (mg/kg/bw/day)

Parameters Control
250 500 1000
Initial body weight (g) 235.1 ± 8.63 230.2 ± 11.62 239.7 ± 11.70 233.0 ± 8.52

PT
Final body weight (g) 297.1 ± 13.42 291.7 ± 14.81 304.8 ± 12.32 299.8 ± 14.64
Corrected final body weight (g) 243.2 ± 3.928 236.7 ± 4.524 245.6 ± 3.854 240.3 ± 3.160
Maternal body weight gain (g) 8.000 ± 4.099 7.500 ± 2.432 5.857 ± 2.807 7.286 ± 1.149

RI
Water intake (mL/day) 22.50 ± 1.443 21.25 ± 2.394 21.25 ± 1.250 22.50 ± 1.443
Food intake (g/day) 29.75 ± 3.198 29.75 ± 3.368 30.25 ± 3.860 30.50 ± 4.481

SC
Corrected final body weight: final body weight minus gravid uterus.
Maternal body weight gain: corrected final body weight minus initial body weight.
Data are expressed as mean ± SEM, n= 6-7 animals/group.

U
The values do not differ from one another at the level of 5% (ANOVA/Tukey test).
AN
M
D
TE
C EP
AC

Figure 1. Evaluation of weight gain of the female rats treated orally with the oil of Caryocar brasiliense
during the 6th until the 15th day of gestation. The data are expressed as mean ± SEM, n= 6-7
animals/group. The values do not differ from one another at the level of 5% (ANOVA/Tukey test).
 26
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Table 2
Absolute and relative organ weight of the female rats treated orally with the oil of Caryocar brasiliense
during the 6th until the 15th day of gestation.

Caryocar brasiliense (mg/kg/bw/day)

Parameters Control
250 500 1000
(g) 0.817 ± 0.04 0.779 ± 0.021 0.785 ± 0.019 0.775 ± 0.031
Heart

PT
(g/100g) 0.276 ± 0.018 0.267 ± 0.009 0.257 ± 0.006 0.259 ± 0.012
(g) 1.474 ± 0.114 1.587 ± 0.102 1.557 ± 0.084 1.577 ± 0.109
Lung
(g/100g) 0.497 ± 0.041 0.544 ± 0.034 0.510 ± 0.026 0.524 ± 0.031

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(g) 0.580 ± 0.026 0.606 ± 0.065 0.614 ± 0.019 0.595 ± 0.021
Spleen
(g/100g) 0.195 ± 0.010 0.207 ± 0.020 0.201 ± 0.006 0.199 ± 0.008

SC
(g) 0.809 ± 0.024 0.844 ± 0.040 0.829 ± 0.022 0.807 ± 0.024
Kidney
(g/100g) 0.273 ± 0.012 0.289 ± 0.012 0.271 ± 0.005 0.269 ± 0.009
(g) 11.14 ± 0.258 11.15 ± 0.284 11.53 ± 0.234 11.52 ± 0.179

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Liver
(g/100g) 3.749 ± 0.051 3.823 ± 0.064 3.784 ± 0.062 3.843 ± 0.040
AN
Data are expressed as mean ± SEM, n= 6-7 animals/group.
The values do not differ from one another at the level of 5% (ANOVA/Tukey test).
M

Table 3
Biochemical parameters in blood of the female rats treated orally with the oil of Caryocar brasiliense
during the 6thuntil the 15th day of gestation.
D

Caryocar brasiliense(mg/kg/bw/day)
TE

Biochemical parameters Control

250 500 1000
Alanine aminotransferase (U/L) 39.27 ± 1.603 39.17 ± 1.995 40.41 ± 1.874 40.79 ± 1.272
EP

Aspartate aminotransferase (U/L) 79.55 ± 4.128 100.9 ± 12.65 85.97 ± 5.632 83.81 ± 4.669
Direct bilirubin (mg/dL) 0.035 ± 0.004 0.034 ± 0.005 0.028 ± 0.004 0.035 ± 0.002
Indirect bilirubin (mg/dL) 0.030 ± 0.008 0.027 ± 0.005 0.030 ± 0.007 0.034 ± 0.009
C

Total bilirubin (mg/dL) 0.066 ± 0.009 0.058 ± 0.006 0.058 ± 0.003 0.071 ± 0.009
Total protein (g/dL) 5.633 ± 0.269 5.757 ± 0.270 5.429 ± 0.119 5.571 ± 0.119
AC

Albumin (g/dL) 3.71 ± 0.29 3.829 ± 0.223 3.600 ± 0.121 3.614 ± 0.137
Globulin (g/dL) 1.917 ± 0.054 1.929 ± 0.064 1.829 ± 0.028 1.971 ± 0.035
Albumin/globulin ratio 2.000 ± 0.223 1.957 ± 0.086 1.943 ± 0.084 1.829 ± 0.110
Blood urea nitrogen (mg/dL) 51.57 ± 3.690 48.46 ± 0.997 53.03 ± 2.569 46.64 ± 2.211
Creatinine (mg/dL) 0.330 ± 0.010 0.330 ± 0.005 0.337 ± 0.012 0.330 ± 0.011
Glucose (mg/dL) 140.9 ± 6.385 143.5 ± 5.213 126.6 ± 5.136 144.3 ± 7.947
Total Cholesterol (mg/dL) 81.75 ± 2.120 84.49 ± 4.194 78.34 ± 3.877 75.80 ± 2.361
Calcium (mg/dL) 11.25 ± 0.226 11.47 ± 0.210 10.97 ± 0.122 11.26 ± 0.168
 27
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Potassium (mmol/L) 5.167 ± 0.214 5.343 ± 0.139 5.257 ± 0.173 5.100 ± 0.109
Sodium (mmol/dL) 138.8 ± 0.391 139.8 ± 0.808 138.8 ± 0.402 138.9 ± 0.436

Data are expressed as mean ± SEM, n= 6-7 animals/group.

The values do not differ from one another at the level of 5% (ANOVA/Tukey test).

Table 4
Hematological parameters in blood of the female rats treated orally with the oil of Caryocar brasiliense

PT
during the 6th until the 15th day of gestation.

Caryocar brasiliense (mg/kg/bw/day)

RI
Hematological parameters Control
250 500 1000
Leukocytes (103/µL) 5.858 ± 0.420 5.713 ± 0.186 5.446 ± 0.480 5.689 ± 0.580

SC
6
Erythrocytes (10 /µL) 5.950 ± 0.159 6.091 ± 0.217 5.909 ± 0.138 5.857 ± 0.104
Hemoglobin (g/dL) 10.72 ± 0.340 10.86 ± 0.346 10.53 ± 0.247 10.44 ± 0.167
Hematocrit (%) 34.00 ± 1.301 34.03 ± 1.210 33.29 ± 0.743 32.71 ± 0.620

U
Mean corpuscular volume (fL) 57.10 ± 0.628 55.87 ± 0.656 56.34 ± 0.155 55.83 ± 0.309
AN
Mean corpuscular hemoglobin 18.00 ± 0.198 17.81 ± 0.133 17.81 ± .0159 17.84 ± 0.048
(pg)
Mean corpuscular concentration 31.55 ± 0.310 31.96 ± 0.301 31.63 ± 0.197 31.93 ± 0.152
hemoglobin (g/dL)
M

Platelets (103/µL) 768.2 ± 40.20 795.6 ± 40.58 800.00 ± 19.21 786.3 ± 18.70
Red Cell Distribution Width (%) 12.25 ± 0.301 12.17 ± 0.228 11.73 ± 0.124 11.71 ± 0.055
D

Neutrophils (%) 20.90 ± 1.722 18.77 ± 1.105 17.49 ± 1.319 17.20 ± 1.688
Lymphocytes (%) 73.85 ± 3.041 74.30 ± 0.932 75.17 ± 2.465 76.83 ± 1.181
TE

Monocytes (%) 3.700 ± 1.597 5.500 ± 1.473 5.700 ± 2.156 4.157 ± 1.201
Eosinophils (%) 1.217 ± 0.162 1.029 ± 0.215 1.214 ± 0.128 1.357 ± 0.203
Basophils (%) 0.233 ± 0.042 0.314 ± 0.055 0.342 ± 0.020 0.385 ± 0.055
EP

Neutrophil Band Cells (%) 0.100 ± 0.044 0.085 ± 0.040 0.085 ± 0.059 0.071 ± 0.035

Data are expressed as mean ± SEM, n= 6-7 animals/group.

C

The values do not differ from one another at the level of 5% (ANOVA/Tukey test).
AC

Table 5
Embryo–fetal toxicity related to reproductive findings of the female rats treated orally with the oil of
Caryocar brasiliense during the 6th until the 15th day of gestation.

Caryocar brasiliense (mg/kg/bw/day)

Parameters Control
250 500 1000
Inseminated females (n) 9 9 9 9
Pregnant females (n) 6 7 7 7
1
Rate of full term pregnancy (%) 66.67 ± 16.67 77.78 ± 14.7 77.78 ± 14.7 77.78 ± 14.7
 28
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Corpora lutea (n)2 11.67 ± 0.494 12.71 ± 0.473 13.86 ± 0.508 12.00 ± 0.755
2
Implantations (n) 9.66± 1.453 10.43 ± 1.360 11.57 ± 0.685 11.00 ± 0.845
1
Rate of implant efficiency (%) 82.13 ± 11.24 81.03 ± 9.22 84.14 ± 5.39 91.52 ± 4.09
1
Pre-implantation loss (%) 17.87± 11.24 18.98 ± 9.22 15.86 ± 5.39 8.48 ± 4.09
1
Resorptions late (n) 0.500 ± 0.223 0.142 ± 0.142 0.142 ± 0.142 0
1
Live fetuses (n) 8.667 ± 1.647 9.429 ± 1.131 10.00 ± 0.645 10.00 ± 1.024
1
Dead fetuses (n) 0.166 ± 0.166 0.142 ± 0.142 0 0

PT
1
Post-implantation loss (%) 16.79 ± 10.74 8.08 ± 2.48 13.32 ± 3.44 9.43 ± 5.21
1
Live male fetuses (n) 4.500 ± 1.118 5.429 ± 0.812 5.000 ± 0.786 3.429 ± 0.649

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2
Live female fetuses (n) 4.167 ± 1.078 4.000 ± 0.755 5.000 ± 0.617 6.571 ± 1.212
Sex ratio (male: female) 1: 0.92 1: 0.736 1: 1 1: 1.916
1
Fetal weight (g) 3.787 ± 0.050 3.775 ± 0.053 3.802 ± 0.036 3.773 ± 0.036

SC
Placental weight (g)1 0.891 ± 0.015 0.798 ± 0.011 0.782 ± 0.011 0.819 ± 0.008
1
Placental index 0.217 ± 0.004 0.213 ± 0.003 0.206 ± 0.003 0.218 ± 0.002

U
Rate of full term pregnancy: (pregnant females x 100) / inseminated females.
Rate of implant efficiency: (nº of implantations / nº of corpora lutea) x 100.
AN
Pre-implantation loss: [(nº of corpora lutea – nº of implantation)/nº of corpora lutea] x 100.
Post-implantation loss: [(nº of implantation – nº of live fetuses) / nº of implantation] x 100.
Placental index: each placental weight / respective fetal weigh.
M

Data are expressed as mean ± SEM. The litters were used as the basis for the analysis of fetal variables.
The values do not differ from one another at the level of 5% (1Kruskal–Wallis/Dunns, 2ANOVA/Tukey).
D
TE
C EP
AC

Figure 2. Percentage (%) of newborns classified small (SAP), adequate (AAP) and large (LAP) for age of
pregnancy, from female rats treated with the oil of Caryocar brasiliense during the 6th until the 15th day
of gestation. The values do not differ from one another at the level of 5% (Qui-Square).
 29
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A B

SC
Figure 3. Evaluation of external abnormalities of the newborns of the female rats treated orally with the
oil of Caryocar brasiliense during the 6th until the 15th day of gestation. (A) Newborn of the group 1000
mg/kg presenting bruise. (B) Newborn of the group control presenting developmental delay (on the right).

U
AN
Table 6
Counting of centers of ossification of the newborns of the female rats treated orally with the oil of
Caryocar brasiliense during the 6th until the 15th day of gestation.
M

Maximum Caryocar brasiliense (mg/kg/bw/day)

Centers of ossification Control
* 250 500 1000
D

Cervical vertebrae 7 7.00 ± 0.00 7.00 ± 0.00 7.00 ± 0.00 7.00 ± 0.00
TE

Anterior phalanges 4 2.283 ± 0.378 2.666 ± 0.627 2.479 ± 0.490 3.179 ± 0.147
Metacarpus 4 3.925 ± 0.047 3.723 ± 0.136 3.857 ± 0.142 4.00 ± 0.00
Sternum 6 6.00 ± 0.00 5.447 ± 0.334 5.600 ± 0.400 6.00 ± 0.00
EP

Metatarsus 5 4.217 ± 0.098 4.143 ± 0.221 4.214 ± 0.225 4.517 ± 0.063

Posterior phalanges 4 1.617 ± 0.560 2.443 ± 0.536 1.736 ± 0.555 2.921 ± 0.227
Caudal vertebrae 7 4.292 ± 0.331 3.457 ± 0.469 3.936 ± 0.596 4.371 ± 0.142
C

Total ossification 37 29.33 ± 1.307 28.91 ± 2.226 28.82 ± 2.219 31.98 ± 0.466
AC

*Aliverti et al., 1979

Data are expressed as mean ± SEM. The litters were used as the basis for the analysis of fetal variables.
The values do not differ from one another at the level of 5% (ANOVA/Tukey).
 ACCEPTED MANUSCRIPT

30

Table 7
Summary of major malformations and minor skeletal variations in the newborns of the female rats treated orally with the oil of Caryocar brasiliense during the 6th until
the 15th day of gestation.

PT
RI
Caryocar brasiliense (mg/kg/bw/day)
Skeletal abnormalities Control
250 500 1000

SC
Fetuses (litters) examined (N) 24 (6) 30 (7) 33 (7) 30 (7)
Abnormalities [% (N of fetuses/N of litters) in:
Cranial bones

U
Nasal, Incomplete ossification V 15.83 ± 12.28 18.09 ± 9.93 20.71 ± 13.82 -

AN
Frontal, Unossified area V - - 5.71 ± 5.71 (2/1) -
Parietal, Incomplete ossification V 7.5 ± 4.78 (2/2) - 2.85 ± 2.85 (1/1) 4.75 ± 4.75 (1/1)
Parietal, Unossified area V - 2.85 ± 2.85 (1/1) 11.43 ± 11.43 -

M
Interparietal, Incomplete ossification V 3.33 ± 3.33 (1/1) - 2.85 ± 2.85 (1/1) -
Interparietal, Unossified area V 3.33 ± 3.33 (1/1) 5.71 ± 5.71 (2/1) 14.29 ± 14.29 -

D
Interparietal, Misshapened MF - - 5.71 ± 3.68 (2/2) -
Supraoccipital, Incomplete ossification V 3.33 ± 3.33 (1/1) 4.75 ± 4.75 (1/1) 14.29 ± 14.29 -

TE
Supraoccipital, Misshapened MF 6.66 ± 6.66 (2/1) 10.47 ± 5.21 29.29 ± 9.53 25.47 ± 10.23
Supraoccipital, Bipartite V - - 8.57 ± 8.57 (3/1) -
Fontanel, Enlarged V 7.5 ± 4.78 (2/2) 4.75 ± 4.75 (1/1) 8.57 ± 5.94 (3/2) 4.75 ± 4.75 (1/1)
EP
Vertebrae
Vertebra, Dumbbell V - - 2.85 ± 2.85 (1/1) -
C

Sternebrae
Sternebra, Absent MF - 26.66 ± 15.32 - -
AC

Sternebra, Incomplete ossification V - 2.85 ± 2.85 (1/1) 14.29 ± 14.29 -

Sternebra, Misshapened V - 14.29 ± 11.31 6.42 ± 4.18 (2/2) -
Sternebra, Misaligned V - - 6.42 ± 4.18 (2/2) -
Sternebra, Bipartite V - - 2.85 ± 2.85 (1/1) -
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31

Rib
Rib, Wavy V 10.83 ± 4.90 15.23 ± 10.25 2.85 ± 2.85 (1/1) 7.61 ± 5.12 (2/2)
Rib, Supernumerary V 3.33 ± 3.33 (1/1) 2.85 ± 2.85 (1/1) 22.86 ± 12.14 14.04 ± 6.82

PT
Rib, Absent MF - - 2.85 ± 2.85 (1/1) -
Forelimbs

RI
Humerus, Short MF - - 2.85 ± 2.85 (1/1) -
Ulna, Short MF - - 11.43 ± 11.43 -
Ulna, Bent MF - - 5.71 ± 5.71 (2/1) -

SC
Radius, Short MF - - 11.43 ± 11.43 -
Radius, Bent MF - - 5.71 ± 5.71 (2/1) -

U
Hindlimbs
Ilium, Short MF - - 14.29 ± 14.29 -

AN
Ischium, Short MF - - 14.29 ± 14.29 -
Pubis, Short MF - - 14.29 ± 14.29 -

M
Femur, Short MF - - 14.29 ± 14.29 -
Fibula, Short MF - - 11.43 ± 11.43 -
Tibia, Short MF - - 11.43 ± 11.43 -

D
(4/1)

TE
In the skeletal evaluation, the abnormalities marked with (V) are considered teratological variations while the abnormalities marked with (M) are considered
EP
malformations (Solecki et al., 2001).
Data are expressed as average percentage of affected fetuses per litter ± SEM. Values in parentheses are (number affected fetuses/number of affected litters). The
values do not differ from one another at the level of 5% (Kruskal–Wallis/Dunns).
C
AC
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A B

RI
U SC
AN
C D
M
D
TE
EP

E F
C

Figure 4. Evaluation of skeletal abnormalities of the newborns of the female rats treated orally with the
oil of Caryocar brasiliense during the 6th until the 15th day of gestation. (A) Newborn of the group 250
AC

mg/kg presenting incomplete ossification of bone nasal. (B) Newborn of the group control presenting
misshaped of bone supraoccipital. (C) Newborn of the group 500 mg/kg presenting absent of one rib. (D)
Newborn of the group 1000 mg/kg presenting ribs supernumerary bilateral. (E) Newborn of the group
control presenting ribs wavy. (F) Newborn of the group 500 mg/kg presenting two sternebra bipartite.
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Highlights:

* The toxicological potential of Caryocar brasiliense pulp oil was investigated through

preclinical studies.

* The Caryocar brasiliense pulp oil was administered orally throughout the period of

organogenesis.

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* No maternal, embryotoxic and teratogenic effects was observed for Caryocar

RI
brasiliense pulp oil.

* The NOAEL of Caryocar brasiliense pulp oil were 1g/kg bw/day, the highest dose

SC
tested.

U
AN
M
D
TE
C EP
AC