Soybean oil, epoxidized, acrylate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2012-February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been conducted according to OECD Guideline No. 422 and under GLP.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 422

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Soybean oil, epoxidized, acrylate

EC Number:

294-415-6

EC Name:

Soybean oil, epoxidized, acrylate

Cas Number:

91722-14-4

Molecular formula:

C63H108O15

IUPAC Name:

Soybean oil, epoxidized, acrylate

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): Epoxidized Soybean Oil Acrylate
- Substance type: UVCB
- Physical state: yellow, viscous liquid
- Analytical purity: 100 %
- Lot/batch No.: S702300037
- Expiration date of the lot/batch: 31 December 2012
- Storage condition of test material: room temperature (15-25 oC, below 70 RH%)

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633
- Age at study initiation: young adult rats, approximately 10 weeks old at starting and 12 weeks at mating
- Weight at study initiation: Males: 346 g – 407 g, Females: 224 g - 277 g
- Housing: Type II and/or III polypropylene/polycarbonate
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum,
- Water (e.g. ad libitum): tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4.– 23.1°C
- Humidity (%): 32 - 57%
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 12 November 2012 To: 30 December 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test item formulations were analysed for concentration and homogeneity at Analytical Laboratory of CiToxLAB Hungary Ltd. Top, middle and bottom triplicate samples were taken from test item formulations on 4 occasions, during the first, second and last weeks and approximately midway of the treatment. Two sets to analyse (which were collected in replicates as practical) and one set as a back-up for any confirmatory analyses. Similarly, one sample was taken in duplicate from the vehicle control group 1 solution for concentration measurements.

Details on mating procedure:

Mating began after the animals have attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male of the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred, for up to 5 days. A vaginal smear were prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were caged individually. Mating pairs were clearly identified in the data, mating of siblings was avoided.

Duration of treatment / exposure:

Test item or negative control material treated groups animals were administered the dosing solutions daily on a 7 days/week basis, by oral gavage using a tipped gavage needle attached to a syringe. A constant volume was administered to all animals. The actual volume administered was calculated and adjusted based on each animal’s most recent body weight.

Doses / concentrations

Remarks:

Doses / Concentrations:
62.5, 250 and 1000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

12 animals/sex/dose

Details on study design:

The dose levels and the vehicle were selected based on available data, formulation and analytical trials and information from previous experimental work, including the results of a repeated dose range finding study in the rat (CiToxLAB Hungary Ltd. study code 12/074-220PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. The oral route was selected as it is a possible route of exposure to the test item in humans.

Test item or negative control material treated groups animals were administered the dosing solutions daily on a 7 days/week basis, by oral gavage using a tipped gavage needle attached to a syringe. A constant volume was administered to all animals. The actual volume administered was calculated and adjusted based on each animal’s most recent body weight.

Dosing of both sexes began after an acclimation period (A) of 6 days after the animal arrival; the animals were dosed for 2 weeks before mating, during the mating/post-mating, and were continued up to the day of necropsy.

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination, as no additional mating was considered required. Females were dosed for 14 days pre-mating, for up to 5 days mating period, through gestation and up to the day of necropsy (at least 4 days post-partum dosing). In one female at 1000 mg/kg (no. 4506) the duration of mating period was 9 days, however this female proved to be not pregnant. The day of birth (viz. when parturition was complete) was defined as Day 0 post-partum. Females showing no-evidence of copulation were sacrificed, 25-27 days after the end of the mating period.

Mating began after the animals have attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male of the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred, for up to 5 days. A vaginal smear were prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were caged individually. Mating pairs were clearly identified in the data, mating of siblings was avoided.

All F1 offspring were terminated on Day 4 post-partum; in order to allow for overnight fasting of dams prior to urine collection on PPD5, offspring were euthanized on PND/PPD 4, and the dams on PPD/PND 5.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS:
All animals: Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and end of the working day. General clinical observations were performed daily, after treatment at approximately the same time with minor variations, or in the afternoon (pm) as practical during the working day, as no peak period of effects was noted after dosing during the first days of treatment. All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including onset, degree and duration of signs as applicable.

DETAILED CLINICAL OBSERVATIONS:
More detailed examinations were made once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning (am) or before treatment. These observations were made outside the home cage in a standard arena, at similar times as practical. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards); special attention were directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. No such clinical signs were observed during the study.

NEUROBEHAVIOURAL EXAMINATION
5 males and 5 females/group, “subgroup A”: Assessment of any potential test item related neurotoxicity was performed during the last exposure week (males on Day 24am; females on PPD 3am). In order to avoid hyperthermia of pups, dams were removed from the pups for not more than approximately 30-40 minutes. Selected animal were subjected to the functional observation battery, including qualitative assessment of the grip strength, and to measurements of the landing foot splay and fore/hind grip strength.

To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rats were dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots was measured.

Fore/hind grip strength measurements were conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support; results are tabulated with individual and mean data.

Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed. Parameters such as, but not limited to body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated.

BODY WEIGHT:
All adult animals were weighed with accuracy of 1 g for randomization purposes, then on Day 0, afterwards at least weekly and at termination. Parent females were weighed on gestation Days GD 0, 7, 14 and 20 and on postpartal Days PPD0 (within 24 hours after parturition), PPD4 and PPD5 (before termination). Body weights of the female animals were additionally weighed on gestational Days GD10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.

FOOD CONSUMPTION
Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then at least weekly (see Study Schedule).

OPHTHALMOSCOPIC EXAMINATION:
The fundus of eyes of all animals was examined before treatment. Five male and 5 female Control and High dose animals (“subgroup A”) randomly selected from groups 1 and 4, during the last week of treatment prior to necropsy (males, Day 24pm, females, PPD 3 pm). Mydriasis was produced after instillation of a mydriatic agent (eye drops "Humapent") into the conjunctival sac. The examination was performed using a Gowlland ophthalmoscope. As no ophthalmoscopic alterations were found, no additional examination was performed in other animals.

OBSERVATION OF THE DELIVERY PROCESS, OFFSPRING AND NURSING INSTINCT
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. All observations were recorded as applicable. No evidence of abnormal deliveries was recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy. Dams were observed to record whether they form a nest from the bedding material and cover their new-borns or not. The efficiency of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded as applicable.

Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities.

Observations are reported individually for each adult animal. In addition to the observations on parent animals, the pups (offspring) were monitored for any behavioural changes. Live pups were counted, sexed, weighed individually within 24 hours of parturition (ex. Day 0 or 1 post-natal, PND0 or 1) and on PND4, with accuracy of 0.01g. All the litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination in order to identify the possible cause of death. All observed abnormalities were recorded

HAEMATOLOGY:
All animals selected for blood sampling were fasted (overnight period of food deprivation). 5 males and 5 females/group, “subgroup B”: For terminal blood sampling of animals selected (subgroup B), 3 samples were taken from each scheduled animal: for haematology (in tubes with K3-EDTA as anticoagulant), one sample for blood clotting times (APTT and PT measurements, in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry. Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.-Parameters checked in table on page 22 of the report were examined.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled necropsy.
- Animals fasted: Yes
- How many animals: 5 males and 5 females
- Parameters checked in table on page 23 of the report were examined.

URINALYSIS:
Urine sampling (approximately 16 hours sampling period) was performed prior to necropsy (males on Day 28; females on PND 5). Parameters checked in table on page 24 of the report were examined.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

FETAL EXAMINATIONS
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. All observations were recorded as applicable. No evidence of abnormal deliveries was recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy. Dams were observed to record whether they form a nest from the bedding material and cover their new-borns or not. The efficiency of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded as applicable.

Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
Observations are reported individually for each adult animal. In addition to the observations on parent animals, the pups (offspring) were monitored for any behavioural changes.

PARAMETERS EXAMINED
Live pups were counted, sexed, weighed individually within 24 hours of parturition (ex. Day 0 or 1 post-natal, PND0 or 1) and on PND4, with accuracy of 0.01g. All the litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination in order to identify the possible cause of death. All observed abnormalities were recorded.

Statistics:

Data were collected using the software PROVANTIS v.7, or were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., then tabulated using the software PROVANTIS v.7, Microsoft Office Word and/or Excel, as appropriate. Numerical data obtained during the conduct of the study were subjected as appropriate to calculation of group means and standard deviations.

The statistical evaluation of appropriate data (marked † below) was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.

Indices:

In parental males the following parameters were detemined: number of fertile pairings, number of infertile males, male mating index and male fertility index.

In parental females the following parameters were detemined: number of pairings, number of pregnant females, number of sperm positive, but non-pregnant females, number of non mated females, female mating index, female fertility index, gestation index, duration of pregnancy (days), number of corpora lutea/dams, number of implantations/dams, number of dams with live pups (day 0 and 4), pre-implantation mortality, intrauterine mortality,
total mortality (intra and extra uterine mortality).

In offspring the following parameters were detemined: mean pup body weight (per pup within the group and per litter) on PND 0 and 4, mean pup body weight gain (per litter) between postnatal Days 0-4, number of live births per litter, and number of viable pups per litter on postnatal Days 0 and 4, survival Index of pups on postnatal Days 0 and 4, sex ratio % (on postnatal Days 0 and 4).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the no observed adverse effect level (NOAEL) for Epoxidized Soybean Oil Acrylate for parental/adult and F1 effects is considered to be 1000 mg/kg bw/day.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat study was to obtain information on the toxicity of the test item following repeated daily administration by oral gavage to Wistar rats. The study also included a reproductive/developmental toxicity-screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Day 4 post-partum. The study was conducted according to OECD Guideline No. 422.

Male and female Wistar rats were treated for 2 weeks pre-mating, then during the mating/postmating period, males for 28 days and females throughout gestation period and up to and including postpartum/lactation Day PPD5

Daily administration of Epoxidized Soybean Oil Acrylate by oral gavage to Wistar rats did not result in test item related mortality or clinical adverse effects at daily, weekly or neurological assessment, in ophthalmological changes, or changes in the body weight, food consumption, haematology, coagulation, clinical chemistry, or urinalysis parameters at dose levels of 62.5, 250, or 1000 mg/kg bw/day during the treatment period under the conditions of this study.

 

No test item related, or adverse effects were noted at evaluation of the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD5, under the conditions of this study.There were no adverse effects ascribed to test item administration on the F1 offspring viability, clinical signs, development or at observations following euthanasia. There were no test item-related changes observed in organ weights, at necropsy or at histopathology for the adult animals of either sex.

 

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for Epoxidized Soybean Oil Acrylate for parental/adult and F1 effects is considered to be 1000 mg/kg bw/day.