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Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

An OECD TG #443 as requested in the Compliance Check Decision number: CCH-D-2114340871-51-01/F, was performed - draft report available.

2-Ethylhexyl acrylate - Extended One-Generation Reproduction Toxicity Study in Wistar Rats Administration via the Diet without extension to produce the F2 generation according to OECD TG 443 (ARTF 2023)

2-Ethylhexyl acrylate was administered to groups of 25 male and 25 female healthy young Wistar rats as a homogeneous addition to the food in different concentrations (0, 1500, 5000 and 12500 ppm). F0 animals were treated at least for 10 weeks prior to mating to produce a litter (F1 generation). Mating pairs were from the same dose group. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 2 different cohorts (1A and 1B) which were subjected to specific postweaning examinations.

The study was terminated with the terminal sacrifice of the F1 rearing animals of cohort 1A. Test diets containing 2-Ethylhexyl acrylate were offered continuously throughout the study.

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances.

Food consumption of the F0 parents and F1 rearing animals was determined regularly once weekly over a period of 1 days (except food consumption of the F0 and F1B males was determined after the 10th premating week) and weekly for F0 and F1B females during gestation days (GD) 6-7, 13-14, 19-20 and postnatal days (PND) 3-4, 6-7, 9-10, 13-14, 17-18 and 20-21.

In general, body weights of F0 parents and F1 rearing animals were determined once weekly. However, during gestation and lactation F0 females were weighed on GD 0, 7, 14 and 20 and on PND 1, 4, 7, 10, 14, 18 and 21.

A detailed clinical observation (DCO) was performed in all F0 parents and F1 animals in cohorts 1A and 1B at weekly intervals.

Estrous cycle data were evaluated for F0 over a three-week period prior to mating until evidence of mating occurred. In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A and 1B females for 2 weeks around PND 75. Moreover, the estrous stage of each F0, 1A and 1B female was determined on the day of scheduled sacrifice.

The F1 pups were sexed on the day of birth (PND 0) and were weighed on the first day after birth (PND 1) as well as on PND 4, 7, 14 and 21. Their viability was recorded. At necropsy, all pups were examined macroscopically.

Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind and randomized fashion using a measuring ocular on all live male and female pups on PND 1.

All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13 and were re-examined on PND 20. If nipple/areola anlagen were recorded, all surviving male pups were carefully re-examined one day prior to necropsy. The number of nipple/areola anlagen were counted.

Date of sexual maturation, i.e. day of vaginal opening (females) or balanopreputial separation (males), of all F1 pups selected to become F1 rearing animals (except F2B rearing animals) was recorded.

Urine samples for clinical pathological investigations were withdrawn from 10 selected F0 and cohort 1A animals per sex and group.

Blood samples for clinical pathological investigations were withdrawn from 10 selected F0 and cohort 1A animals per sex and group.

Further blood samples were taken from all surplus (culled) PND 4 pups per sex and group as well as from 10 surplus PND 22 pups per sex and group.

Various sperm parameters (motility, sperm head count, morphology) were assessed in the F0 generation males and cohort 1A males at scheduled sacrifice or after appropriate staining.

All F0 parental animals were assessed by gross pathology (including weight determinations of several organs) and subjected to an extensive histopathological examination; special attention being paid to the organs of the reproductive system. A quantitative assessment of primordial and growing follicles in the ovaries was performed for all control and high-dose F1 rearing females of cohort 1A.

All F1 rearing animals were assessed by different pathological and histopathological examinations. In cohort 1B, histotechnical processing and examination by light microscopy was not performed.

Stability, correct concentrations and homogeneous distribution of 2-Ethylhexyl acrylate in the diet were all confirmed through analytics.

For the male animals the overall mean dose of 2-Ethylhexyl acrylate throughout all study phases and across all cohorts was approx. 119 mg/kg body weight/day (mg/kg bw/d) in the 1500 ppm group, approx. 357 mg/kg bw/d in the 5000 ppm group and approx. 998 mg/kg bw/d in the 12500 ppm group. For the female animals the overall mean dose of 2-Ethylhexyl acrylate throughout all study phases and across all cohorts was approx. 135 mg/kg bw/d in the 1500 ppm group, approx. 453 mg/kg bw/d in the 5000 ppm group and approx. 1136 mg/kg bw/d in the 12500 ppm group.


There were no test substance-related mortalities or adverse clinical observations noted in any of the treatment groups in the F0 parental animals and F1 offspring. In particular, regularly conducted detailed clinical observations revealed no effects.


The high concentration of the test substance (12500 ppm) produced signs of adverse local effects which were subsequently conveyed to some subsequent systemic effects in the F0 parental rats and F1 adolescents.


In the 12500 ppm and partly also 5000 ppm F0 parental animals food consumption was reduced during several episodes of the study. No comparable findings were noted in the F1A and F1B offspring. Although they might be related to the irritating properties of the test item (see pathology), those changes were minor and followed a patchy pattern, thus they did not qualify as adverse test substance-related effects by themselves.


Body weights and body weight change of the 12500 ppm male F0 and F1 rats were consistently and, in many parts of the study significantly, below the concurrent control across all cohorts and study periods, including terminal body weight. Female F0 and F1 rats were less sensitive against this effect, significant body weight decreases were limited to single episodes during the study, overall the pattern and severity of changes pointed toward a borderline outcome.


Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the test compound of 12500 ppm in F0 as well as F1A rats of both sexes.

Regarding pathology, target organs were the glandular stomach and the kidneys.

F0-Generation parental animals
In females of test group 03 (12500 ppm), an increased number of foci was macroscopically detected in the glandular stomach. This finding was correlated to an increased number of erosions/ulcers histologically detected in the glandular stomach and was regarded as treatmentrelated and locally adverse.

In males of test group 03 (12500 ppm), an increased incidence of basophilic tubules was detected in the kidneys. All animals were minimally to mildly affected, in most affected animals basophilic tubules could only be seen unilaterally. No signs of inflammation or degeneration were detected. In combination with increased incidences of transitional epithelial cells and granulated and epithelial casts detected in urinalysis it is assumed that a beginning α2μ -globulinuria could be the cause of the increase in basophilic tubules, even if the a2μ globulinuria was not detected by immunohistochemistry. This finding is regarded as treatment-related but not as adverse.


F1 rearing animals, cohort 1A
In females of test group 13 (12500 ppm), an increased number of erosions/ulcers was histologically detected in the glandular stomach. This finding was regarded as treatment-related and locally adverse.

In males of test group 11, 12, and 13 (12500 ppm), an increased incidence of basophilic tubules was detected in the kidneys. All affected animals displayed minimal to mild basophilic tubules.
In most affected animals basophilic tubules could only be seen in one kidney. No signs of inflammation or degeneration were detected. In combination with the increased incidences transitional epithelial cells (test group 13 only) and granulated and epithelial casts (test groups 12 and 13) detected in urinalysis it is assumed that a beginning α2μ -globulinuria could be the cause of the increase in basophilic tubules. This finding is regarded as treatment-related but not as adverse.


F1 rearing animals, cohort 1B
The statistically significant organ weight increases and decreases were regarded as secondary to the decreased body weight. No histopathological examination was performed.


Surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts)
The statistically significant organ weight increases were regarded as secondary to the decreased body weight. No histopathological examination was performed.


All other macroscopic or microscopic findings occurred either individually or were biologically equally distributed about control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.


There were no indications from clinical examinations as well as gross and histopathology, that 2-Ethylhexyl acrylate adversely affected the fertility or reproductive performance of the F0 parental animals up to and including the administered high concentration of 12500 ppm. Estrous cycle data, sperm quality of males, mating behavior, conception, gestation, parturition, lactation and weaning as well as sexual organ weights and gross and histopathological findings of these organs (specifically the differential ovarian follicle count) were comparable between the rats of all dose groups and control and/or ranged within the historical control data of the test facility.


A concentration of 12500 ppm was associated with statistically significantly reduced numbers
of implants, which subsequently caused a smaller average litter size in F1 offspring of the highdose group (both within historical control range). However, all reproductive characteristics listed above remained unchanged and no increase in intrauterine mortality was noted in the F0 parents of this offspring. Therefore, it is likely that the relatively high implantation number and litter size (in the upper historical range) in control caused a statistical difference to the highdose number which is rather normal for the rat strain used in this study. Overall, no toxicological relevance is assumed for this apparent finding.


For all liveborn male and female pups of the F0 parents, no test substance-induced signs of developmental toxicity were noted at concentration levels as high as 12500 ppm. Postnatal survival as well as post-weaning development of the offspring in all treatment groups until puberty remained unaffected by the test substance. Furthermore, clinical and/or gross necropsy examinations of these F1 pups revealed no adverse findings.


Pup body weights did not show significant differences between control and all treatment groups. However, mean pup weights were slightly lower in the high-dose group than in the control group (by about 5%) at the end of lactation, though the difference did not become statistically significant. This minor change reflects the decrease in high-dose pup body weight gain towards the end of lactation.


Likewise, there was no significant difference in mean pup body weight change noted between control and test groups 01 and 02 (1500 and 5000 ppm). Mean pup body weight change of the high-dose animals was also comparable to the control group throughout the first two weeks of lactation but was statistically significantly below the concurrent control during the last week of lactation. During the last week of lactation, the offspring already consume considerable amounts of medicated diet in addition to breast milk feeding and the post-weaning body weight gain of the male high-dose adolescents in the F1A and F1 B cohorts continued to be lower compared to the control. Thus, it can be assumed that the slightly lower pre-weaning body weight gain in the high-dose group was caused by the combined diet and milk exposure of the offspring to the test compound, which is likely to be higher than exposure by either source alone. However, the pup body weight gain effects had no influence on postnatal pup survival or well-being, neither during late lactation nor later, as clinical and/or gross necropsy examinations of the high-dose F1 pups revealed no adverse findings.


Measurement of thyroid hormones revealed no consistent adverse effects caused by the test substance in the F0 and F1 adult animals as well as the F1 preweaning offspring.


Neither the anogenital distance/index nor the percentage of male pups showing presence of nipples/areolas, both very sensitive marker of potential endocrine-mediated imbalances, revealed any test substance-related effects.


A statistically significant delay in vaginal opening of about one day beyond the concurrent control was observed in the female F1 offspring of the high-dose group (12500 ppm). The delay is within the historical control range of the test facility. There was a mild effect on post-reaning body weight development in the affected females around the timing of puberty (around study day 7 in the F1A cohort) notable. On the other hand, there was no effect on estrous cyclicity or the integrity of sexual organs in the investigated F1 females, including differential ovarian follicle count, during later life. Thus, there is evidence that this finding is due to an unspecific minimal delay of general development rather than a specific effect on the timing of puberty. Overall, it is regarded as of no toxicological relevance.


Neither statistically significant nor toxicologically relevant effects of the test substance on the timing of preputial separation was noted in any of the treatment groups. In addition, no effect on the integrity of sexual organs (including accessory sexual glands) as well as on sperm quality was noted in all investigated F1 males.


There was no evidence that the test substance produced any developmental immunotoxicity. Absolute and relative lymphocyte subpopulation cell counts in the spleen tissue (B-, Tlymphocytes, CD4-, CD8-T-lymphocytes and natural killer (NK) cells) displayed no treatmentrelated changes.

CONCLUSION

Thus, under the conditions of the present extended one-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 5000 ppm (about 357 mg/kg bw/d in males, 453 mg/kg bw/d in females) in the F0 parental and the F1 adolescent/adult rats, based on evidence for local toxicity in the gastrointestinal tract which conveyed to systemic toxicity such as decreased body weight/body weight gain across gener-

ations and cohorts, at the LOAEL (Lowest Observed Adverse Effect Level) of 12500 ppm (about 998 mg/kg bw/d in males, 1136 mg/kg bw/d in females).

The NOAEL for fertility and reproductive performance is 12500 ppm (about 998 mg/kg bw/d in males, 1136 mg/kg bw/d in females), the highest dose tested

The NOAEL for developmental toxicity in the F1 progeny is 12500 ppm (about 998 mg/kg bw/d in males, 1136 mg/kg bw/d in females), the highest dose tested.

The NOAEL for developmental immunotoxicity for the F1 progeny is 12500 ppm (about 998 mg/kg bw/d in males, 1136 mg/kg bw/d in females), the highest dose tested.

As described in the section “Toxicokinetics, metabolism and distribution (ADME)” of the dossier, 2-ethylhexyl acrylate is rapidly and extensively absorbed, distributed and eliminated after oral administration. In vitro and in vivo studies have demonstrated that 2-EHA undergo carboxylesterase-catalyzed hydrolysis to acrylic acid and 2-ethylhexanol. The hydrolysis is so fast that exposure to the parent compound is considered to be not significant. Therefore, the reproductive toxicity of 2-EHA can also further be evaluated from studies performed on the substance itself and its degradation products, acrylic acid (AA) and 2-ethylhexanol (2-EH) and also from a substance, n-butyl acrylate (n-BA), belonging to the same chemical category.

The toxicity to reproduction (effects on fertility and developmental toxicity) of 2-EH was evaluated as a part of the CORAP process and it was concluded that the available data does not raise concern that 2-EH would affect fertility and sexual function. The reproductive toxicity of AA was evaluated in an OECD TG# 416 study and no effects on the reproductive function was observed up to the top dose level of 460 mg/kg bw/d.

In addition, with structural analog n-Butyl acrylate, no reproductive toxicity was observed up to the top dose level of 150 mg/kg/day in an OECD TG #443 study and up to the top dose level of 400 mg/kg/d in a range-finding study performed following a protocol derived from the OECD TG# 421.

Link to relevant study records

Referenceopen allclose all

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Study period:
19 Mar 2021 to 2023
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
Due to unscheduled delays in the contract laboratory, the final report for this study is not yet available, so only the draft report is available so far. However, another dossier update including the final study results and an assessment of its impact on the exposure assessment will be done as soon as possible.



Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
25 Jun 2018
GLP compliance:
yes
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS

- Premating exposure duration for parental (P0) animals : Standard design, 10 weeks
- Basis for dose level selection : preliminary range-finding study will be conducted beforehand
- Exclusion of extension of Cohort 1B: exclusion
- Termination time for F2 : -
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B
- Exclusion of developmental immunotoxicity Cohort 3
- Route of administration : oral
see "justtification for type of information" for more details
Specific details on test material used for the study:
2-Ethylhexyl acrylate purity 99.8 %
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 34 days
- Housing: 5 animal (Polysulfonate cages Typ 2000P (H-Temp):During premating, male animals after mating (post-mating), females after weaning and animals of cohorts 1A and 1B); 1 animal (Polycarbonate cages type III During mating, gestation, lactation) Exceptions: During mating: 1 male/1 female per cage; During lactation, rearing up to weaning: 1 dam with her litter
- Diet (e.g. ad libitum): Mouse and rat maintenance diet “GLP”, Granovit AG, Kaiseraugst, Switzerland; ad libitum
- Water (e.g. ad libitum): drinking water, ad libitum
- Acclimation period: 7 days before administration, during the acclimatization period, the animals will become accustomed to the environmental conditions and to the diet.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Details on exposure:
Due to the weaknesses in the OECD422 based on technical difficulties, especially the preparation of the test substance in the application media (concentration analysis showed large variabilities and dosing formulations did not meet the acceptability criteria), the study is of limited reliability. To avoid these difficulties in requested OECD 443 including range-finding study it was decided to perform those tests by dietary route. Since 2EHA is an irritant monomer the local inflammatory effects in the stomach might also be reduced by application of the test substance via the diet.

PREPARATION OF DOSING SOLUTIONS:
Preparation frequency:
The test substance preparations is prepared at intervals which guarantee that the test substance concentrations in the diet will remain stable.
Storage conditions of the preparations:
The test-substance preparations are split in aliquots and stored at -18°C in order to offer new test-substance preparations. The food in the food hoppers are changed at least every three days (after the preparations have reached room temperature).

Details on mating procedure:
In general, each of the male and female animals was mated overnight at a 1 : 1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group.
The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 06.30 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was
denoted " gestation day (GD) 0" and the following day "gestation day (GD) 1".
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in the diet for a period of 32 days in freezer following 4 days at room temperature was verified before the start of the study.
Analyses were carried out 4 times during the study. At the beginning, in the middle and toward the end of the study, as well as for female diets once in the lactation period (Concentration control (mid dose) and Homogeneity analyses (low + high dose)).
Duration of treatment / exposure:
The F0 animals, with the exception of the controls, were fed diets supplying the appropriate amounts of the test substance for approximately 10 weeks prior to breeding and continuing through breeding (up to two weeks), and for a maximum of 4 post-mating weeks (males) or gestation (three weeks) and lactation (three weeks) for females. Selected F1 offspring (cohorts 1A and 1B) were maintained on the test diet until one day before sacrifice.
Frequency of treatment:
daily
Details on study schedule:
F0 generation animals and their progeny:

The male and female rats were about 27 (±1) days old when they arrived from the breeder. During an acclimatization period of about 7 days, animals with lowest and highest body weights were eliminated from the study and used for other purposes. The 100 male and 100 female animals required for the study were about 34 (±1) days old at the beginning of treatment and their weight variation did not exceed 20 percent of the mean weight of each sex.

The assignment of the animals to the different test groups was carried out using a randomization program, according to their weight one day before the beginning of the administration period (day -1).

After the acclimatization period, the test substance was administered to the parental animals as addition to the diet continuously throughout the entire study. The animals of the control group were treated in the same way, with the vehicle (diet only). Treatment ended about 16 hours before sacrifice.

After a minimum of 10 weeks after the beginning of treatment, males and females from the same dose group were mated, overnight at a ratio of 1 : 1

The females were allowed to deliver and rear their pups (F1 generation pups) until PND 4 (standardization) or PND 21 or 22 (depending on the cohort). Pups of the F1 litter were selected (F1 rearing animals) and assigned to 2 different cohorts which were subjected to specific post-weaning examinations.

On PND 4 blood samples were collected from all surplus (culled) F1 pups per sex and group.
On PND 22 blood samples were collected from 10 surplus F1 pups per sex and group.

Blood samples were taken from 10 animals per test group of the F0 parental animals and cohort 1A animals.
Shortly before weaning of the F1 pups the F0 generation parental male animals were sacrificed. After weaning of F1 pups the F0 generation parental female animals were sacrificed.


F1 pups and selection of cohorts:

Before weaning of the F1 generation pups on PND 21, 45 male and 45 females per group were randomly selected, to be placed into cohorts. Obvious runts (those pups whose body weight was > 25% below the mean body weight of the control group, separate for sexes) were not included.

Cohorts:
Cohort 1A: One male and one female/litter (20/sex/group)
Cohort 1B: One male and one female/litter (25/sex/group)
Dose / conc.:
1 500 ppm
Remarks:
overall mean dose: male: 119 mg/kg bw/d + female 135 mg/kg bw/d
Dose / conc.:
5 000 ppm
Remarks:
overall mean dose: male: 357mg/kg bw/d + female 453 mg/kg bw/d
Dose / conc.:
12 500 ppm
Remarks:
overall mean dose: male: 998 mg/kg bw/d + female 1136 mg/kg bw/d
During lactation period, concentration in the diest of the F0 will be reduced to 50%.
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a modified 421 study (reproduction/developmental toxicity screening test, project No. 81R0903/00S059), 2-Ethylhexyl acrylate was administered to each 10 male and female Wistar rats at concentrations in the diet of 0, 3000, 7500 and 15000 ppm. During lactation, concentrations in the diet of the F0 females were reduced to 50%. In the highest test group (15000 ppm), mean food consumption and body weight development were the most affected parameters.
Food consumption was (statistically significantly) reduced in males (up to 20.2% below control) and females (up to 14.5% below control) of test group 3 and in males of test group 2 (up to 18% below control) during premating. During gestation, females of test group 3 recovered to values comparable to control whereas during lactation, mean food consumption showed a higher variation.
Body weight (change) was decreased in males of test group 3 with a body weight loss in the beginning of the study (body weight change during study days 0-7: -2 g versus 12.6 g in control). However, these males recovered and gained weight towards the end of the study (4.8% below control). For females of test group 3, body weight (change) was not affected during premating but showed a statistically significant decrease during gestation (8.4% below control). During lactation, body weight of these animals recovered almost to control values.
In this extended one-generation study, pretreatment/premating will be prolonged to 10 weeks and animals are younger compared to the 421-study leading to a higher substance intake in the beginning of the study. In this study, the highest concentration in the diet aims to reach limit dose considering all the different study phases.
Based on the above-mentioned results and at the request of the sponsor, the following concentrations in the diet were selected:
1500* ppm as low concentration
5000* ppm as mid concentration
12500* ppm as high concentration
*During the lactation period the 2-Ethylhexyl acrylate concentrations in the diet of the F0 was reduced to 50%. This dietary adjustment derived from historical body weight and food consumption data will maintain the dams at the desired target doses of 2-Ethylhexyl acrylate during this period of increased food intake.

- Other:
F0 generation parental animals and F1 pups:
Male and female animals, aged about 4 weeks when supplied, will be used as F0 generation parental animals. After an acclimatization period of at least 7 days, these animals will be tended to for at least 10 weeks.
Then the F0 animals will be mated. The female F0 animals will be allowed to deliver and rear their pups (F1 generation pups) until postnatal days (PND) 4 or 21. The male F0 generation parental animals will be sacrificed during rearing (see 2.5. time schedule). The female F0 generation parental animals will be sacrificed after weaning of the F1 generation pups. All F0 females will be sacrificed after weaning.

F1 pups and selection of cohorts:
Before weaning of the F1 generation pups on PND 21, 45 males and 45 females per group were randomly selected (selection see below), to be placed into cohorts.. Obvious runts (those pups whose body weight is equal to or greater than 25% below the mean body weight of the control group, separate for sexes) will not be included.
Cohorts:
Cohort 1A: One male and one female/litter (20/sex/group)
Cohort 1B: One male and one female/litter (25/sex/group)

Standardization of litters (culling) of F1 generation:
On PND 4, all litters were standardized to 10 pups per litter. Whenever possible, each litter contains 5 male and 5 female pups; as a rule, the first 5 surviving pups/sex in each litter were used.
Parental animals: Observations and examinations:
MORTALITY:
A check for moribund and dead animals was made twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays.

CAGE SIDE OBSERVATIONS: (for F0 generation parental animals and selected F1 rearing animals)
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. If such signs occur, the animals will be examined several times daily. Abnormalities and changes will be documented for each animal.
The parturition and lactation behavior of the dams was generally evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver or umbilical cord not cut) will be documented on an individual dam basis.
On weekdays (except Saturdays, Sundays and public holidays) the parturition behavior of the dams will be inspected in the afternoons in addition to the evaluations in the mornings.
The day of parturition is considered to be the 24-hour period from about 15:00 h of one day until about 15:00 h of the following day. Departures from this procedure may occur on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS:
All F0 parental animals and F1 animals in cohorts 1A and 1B were subjected to detailed clinical observations (DCO) outside their cages once before the beginning of the administration period (day 0 only for F0 parental animals) and subsequently once per week (in the morning). For observation, the animals will therefore be removed from their cages and placed in a standard arena (50 × 37.5 × 25 cm).
The scope of examinations and the scoring of the findings observed will be based on the current index of findings in ToxLIMS software and includes but is not limited to the following parameters listed:
Abnormal behavior in handling, Fur, Skin, Posture, Salivation, Respiration, Activity/arousal level, Tremors, Convulsions, Abnormal movements, Gait abnormalities, Lacrimation, Palpebral closure, Exophthalmos, Assessment of the feces discharged during the examination (appearance/consistency), Assessment of the urine discharged during the examination, Pupil size

BODY WEIGHT:
In general, the body weight of the male and female F0 parental animals and F1 rearing animals was determined once a week at the same time of the day (in the morning), if possible.
The following exceptions are notable for the female parental animals:
During the mating period of the F0 parental animals, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
Females with litter were weighed on the day after parturition (PND 1) and on 4, 7, 10, 14, 18 and 21.
Females without litter and after weaning (PND 21) were weighed once a week

FOOD CONSUMPTION
Generally, food consumption was determined once a week (over a period of 7 days) for male and female F0 parental animals and F1 rearing animals, with the following exceptions:
•Food consumption was not determined after the 10th premating week (male F0 and F1B animals) and during the mating period (male and female F0 parental animals).
• Food consumption of the F0 females with evidence of sperm was determined weekly for GD 6-7, 13-14 and 19-20.
• Food consumption of the F0 females, which gave birth to a litter was determined for PND 3-4, 6-7, 9-10, 13-14, 17-18 and 20-21.
• Food consumption of females showing no positive evidence of sperm in the vaginal smear was determined once a week during this mating interval as were the males
• Food consumption of females without litter and after weaning (PND 21) was determined once a week


WATER CONSUMPTION AND COMPOUND INTAKE:
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

DETAILED CLINICAL OBSERVATION DCO)
Detailed clinical observations were performed in all F0 parental animals once before the administration and supsequently once per week and in cohorts 1A and 1B at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable.
For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 × 37.5 × 25 cm). The following parameters listed were assessed:
1. Abnormal behavior in handling
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos (Protruding eyeball)
15. Assessment of the feces excreted during the examination (appearance/consistency)
16. Assessment of the urine excreted during the examination
17. Pupil size

CLINICAL PATHOLOGY:
Clinical Pathology in F0 parental and cohort 1A animals
Samples were withdrawn from the first 10 surviving F0 parental (females with litter, corresponding males) and the first 10 surviving cohort 1A males and females per group at termination. Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood examinations were carried out in a randomized sequence. The list of randomization instructions was compiled with a computer.
In the afternoon preceding the day of urinalysis, the animals will be individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens will be examined in a randomized sequence (the list of randomization instructions will be compiled with a computer).

The following parameters were examined:

Hematology:
Leukocytes, Erythrocytes, Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelets, Differential blood count, Reticulocytes,
Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument.
Clotting tests were carried out using a ball coagulometer.
Prothrombin time (Hepato Quick’s test) (HQT)

Clinical chemistry:
Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Serum g-glutamyl transferase, Sodium, Potassium, Chloride, Inorg. phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins, Triglycerides, Cholesterol

Hormone evaluations:
T4 (thyroxine), THS

Splenic lymphocyte subpopulation analysis:
Ten males and females per group of cohort 1A were used to perform a splenic lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells) using one half of the spleen, the other half of the spleen being preserved for histopathological evaluation.

Urinalysis:
Volume, Color, Turbidity, pH value, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Microscopy of sediment
Oestrous cyclicity (parental animals):
Estrous cycle determinations
Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 parental rats for a minimum of 3 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation.
In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A and 1B females for 2 weeks around PND 75.
At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female and cohort 1A and 1B female with scheduled sacrifice.
Sperm parameters (parental animals):
Sperm parameters
After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male F0 parental animals and all cohort 1A males sacrificed on schedule:
Sperm motility examinations were carried out in a randomized sequence.
Parameters and methods:
Sperm motility (MO-TILE_C); % , microscopic evaluation
Sperm morphology (AB-NORMAL5_C); %, vital staining with eosin; microscopic evaluation
Sperm head count(cauda epididymis) (TS/gC), Mio/g cauda epididymis, microscopic evaluation with MAKLER chamber after homogenization
Toxicity of 2-Methoxyethanol Applied Dermally to Occluded and Nonoc-
Sperm head count (testis) (TS/gT), Mio/g testis, microscopic evaluation with MAKLER chamber after homogenization
Litter observations:
CLINICAL EXAMINATIONS (of F1 generation pups)
Pup status and litter size after birth:
The status (sex, live-born or stillborn) and number of all pups delivered from the F0 parents will be determined as soon as possible after birth. At the same time, the pups will also be examined for gross-morphological changes.
Pup viability/mortality:
In general, a check will be made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays.
Pups, which died before the first determination of their status on the day of birth, will be defined as stillborn pups.
Clinical signs:
All live pups will be examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups show particular findings, these will be documented for each pup.
Body weights:
The pups will be weighed on the day after birth (PND 1) and on PND 4, 7, 14 and 21. Whenever possible, the individual weights will always be determined at the same time of the day (in the morning) and on PND 4 before standardization of the litters. Additionally, the body weight of all F1 rearing animals will be determined on the day of vaginal opening and preputial separation.
Anogenital distance:
Anogenital distance (defined as the distance from the center of the anal opening to the base of the genital tubercle) measurements will be conducted in a blind randomized fashion, using a measuring ocular, on all live pups on PND 1.
Anogenital index:
The anogenital index will be calculated according to the following formula: anogenital distance [mm]/ anogenital index = cubic root of pup weight [g]
Nipple/areola anlagen:
All surviving male pups will be examined for the presence of nipple/areola anlagen on PND 13 and again towards the end (either PND 20 or 21) of the lactation phase. The number of nipple/areola anlagen will be counted.
Sexual maturity:
Developmental landmarks of sexual maturity of all F1 rearing animals (cohort 1A and 1B) will be recorded on the following days: From PND 27 onward: opening of the vagina; from PND 38 onward: balanopreputial separation. Recording will be completed when all animals are sexually mature or latest on PND 66 in males and in females.
Hormones in PND 4 and 22 F1-offspring:
Blood sampling: Blood samples will be withdrawn from all surplus (culled) PND 4 pups. PND 4 samples will be pooled per sex and litter. If not enough pools per sex are sufficient for the hormone measurements, samples will be pooled regardless of sex per litter.
Blood samples will be withdrawn from 10 surplus PND 22 offspring per sex and group (as far as possible 1 male or 1 female of different litters). The blood samples will be collected after decapitation (following isoflurane anesthesia) from the Vena cava cranialis.
Hormone evaluations:
The following hormones will be determined in the serum samples: T4 (thyroxine); TSH
Postmortem examinations (parental animals):
(F0 generation parental animals and cohort 1A)

Necropsy
All F0 parental animals and all cohort 1A animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights:
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (final body weight), Adrenal glands (fixed), Brain, Caudae epididymides, Epididymides, Heart, Kidneys, Liver, Lymph nodes, axillary (10 animals per sex per group, cohort 1A animals only), Lymph nodes, mesenteric (10 animals per sex per group, cohort 1A animals only), Ovaries, Pituitary gland (fixed), Prostate (ventral and dorsolateral part together, fixed), Testes, Seminal vesicles including coagulating glands (fixed), Spleen, Thymus (fixed), Thyroid glands (with parathyroid glands) (fixed), Uterus with cervix.
All paired organs will be weighed together (left and right).

Organ/Tissue fixation:
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Adrenal glands, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymis, left (modified Davidson’s solution), Esophagus, Eyes with optic nerve (modified Davidson’s solution), Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Liver, Lungs, Lymph nodes, axillary, Lymph nodes, mesenteric, Mammary gland (male and female), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Pituitary gland, Prostate, Rectum, Sciatic nerve, Seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), Spleen, Stomach (forestomach and glandular stomach), Target organs, Testis, left (fixed in modified Davidson ´s solution), Thymus, Thyroid glands (with parathyroid glands), Trachea, Urinary bladder, Uterus, Vagina, Vas deferens.

The left testis and left epididymis of all male F0 parental and Cohort 1A animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters analysis .
In case of macroscopic findings in the right testis or right epididymis, this testis as well as the corresponding epididymis were fixed for histopathological examination and the left testis and epididymis were used for sperm analysis.

The uteri of all cohabited female F0 parental animals were examined for the presence and number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method). Then the uteri were rinsed carefully in physiologic salt solution (0.9 % NaCl). When the examinations were completed, the uteri were transferred to the Pathology Laboratory for further processing.
Spleens of 10 animals per sex per group of cohort 1A were split in two comparable parts (transversally). One part of the spleen was fixed in 4% neutral buffered formaldehyde and afterwards embedded in paraplast. The other part of the spleen was frozen at -80o C, being used to perform a splenic lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells).


Histopathology:
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below:
All gross lesions, Adrenal glands, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymis, left (modified Davidson’s solution), Esophagus, Eyes with optic nerve (modified Davidson’s solution), Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Liver, Lungs, Lymph nodes, axillary, Lymph nodes, mesenteric, Mammary gland (male and female), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyriod glands, Pituitary gland, Prostate, Rectum, Sciatic nerve, Seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), Spleen, Stomach (forestomach and glandular stomach), Target organs, Testis, left (fixed in modified Davidson ´s solution), Thymus, Thyroid glands (with parathyroid glands), Trachea, Urinary bladder, Uterus, Vagina, Vas deferens.

The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004).
A correlation between gross lesions and histopathological findings was attempted.
Special attention was given to stages of spermatogenesis in the male gonads.
Special attention was also given to the synchrony of the morphology in ovaries, uterus, cervix, and vagina to the estrous cycle status.
An immunohistochemial stain for a2μ in the kidneys was performed exemplarily in 2 control and 2 high dose male animals of F0 animals (animal no. 2, 13, 77, 92).
Reproductive organs of all F0 animals suspected of reduced fertility were subjected to histopathological investigation.
A differential ovarian follicle count (DOFC) was conducted in test groups 10 and 13 (Cohort 1A females) according to Plowchalk et.al. (1993).

Differential Ovarian Follicle Count (DOFC) in cohort 1A females
A differential ovarian follicle count (DOFC) was conducted in test groups 10 and 13 (cohort 1A females) according to Plowchalk et.al. (1993) by the Test Facility. In general, sections were prepared with 2 - 3 μm thickness and serial sections were taken every 100 μm to complete about 20 cut levels across the whole ovarian tissue. For the counting of primordial and growing follicles, HE-stained slides were prepared from all cut levels. Counting was performed on slides digitalized with a Hamamatsu NanoZoomer 2.0 slide scanner using the Hamamatsu viewing software (NDP.view).
Postmortem examinations (offspring):
(F0 generation parental animals and cohort 1A)

Necropsy
All F0 parental animals and all cohort 1A animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights:
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (final body weight), Adrenal glands (fixed), Brain, Caudae epididymides, Epididymides, Heart, Kidneys, Liver, Lymph nodes, axillary (10 animals per sex per group, cohort 1A animals only), Lymph nodes, mesenteric (10 animals per sex per group, cohort 1A animals only), Ovaries, Pituitary gland (fixed), Prostate (ventral and dorsolateral part together, fixed), Testes, Seminal vesicles including coagulating glands (fixed), Spleen, Thymus (fixed), Thyroid glands (with parathyroid glands) (fixed), Uterus with cervix.
All paired organs will be weighed together (left and right).

Organ/Tissue fixation:
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Adrenal glands, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymis, left (modified Davidson’s solution), Esophagus, Eyes with optic nerve (modified Davidson’s solution), Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Liver, Lungs, Lymph nodes, axillary, Lymph nodes, mesenteric, Mammary gland (male and female), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Pituitary gland, Prostate, Rectum, Sciatic nerve, Seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), Spleen, Stomach (forestomach and glandular stomach), Target organs, Testis, left (fixed in modified Davidson ´s solution), Thymus, Thyroid glands (with parathyroid glands), Trachea, Urinary bladder, Uterus, Vagina, Vas deferens.

The left testis and left epididymis of all male F0 parental and Cohort 1A animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters analysis .
In case of macroscopic findings in the right testis or right epididymis, this testis as well as the corresponding epididymis were fixed for histopathological examination and the left testis and epididymis were used for sperm analysis.

The uteri of all cohabited female F0 parental animals were examined for the presence and number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method). Then the uteri were rinsed carefully in physiologic salt solution (0.9 % NaCl). When the examinations were completed, the uteri were transferred to the Pathology Laboratory for further processing.
Spleens of 10 animals per sex per group of cohort 1A were split in two comparable parts (transversally). One part of the spleen was fixed in 4% neutral buffered formaldehyde and afterwards embedded in paraplast. The other part of the spleen was frozen at -80o C, being used to perform a splenic lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells).


Histopathology:
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below:
All gross lesions, Adrenal glands, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymis, left (modified Davidson’s solution), Esophagus, Eyes with optic nerve (modified Davidson’s solution), Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Liver, Lungs, Lymph nodes, axillary, Lymph nodes, mesenteric, Mammary gland (male and female), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyriod glands, Pituitary gland, Prostate, Rectum, Sciatic nerve, Seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), Spleen, Stomach (forestomach and glandular stomach), Target organs, Testis, left (fixed in modified Davidson ´s solution), Thymus, Thyroid glands (with parathyroid glands), Trachea, Urinary bladder, Uterus, Vagina, Vas deferens.

The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004).
A correlation between gross lesions and histopathological findings was attempted.
Special attention was given to stages of spermatogenesis in the male gonads.
Special attention was also given to the synchrony of the morphology in ovaries, uterus, cervix, and vagina to the estrous cycle status.
An immunohistochemial stain for a2μ in the kidneys was performed exemplarily in 2 control and 2 high dose male animals of F0 animals (animal no. 2, 13, 77, 92).
Reproductive organs of all F0 animals suspected of reduced fertility were subjected to histopathological investigation.
A differential ovarian follicle count (DOFC) was conducted in test groups 10 and 13 (Cohort 1A females) according to Plowchalk et.al. (1993).

Differential Ovarian Follicle Count (DOFC) in cohort 1A females
A differential ovarian follicle count (DOFC) was conducted in test groups 10 and 13 (cohort 1A females) according to Plowchalk et.al. (1993) by the Test Facility. In general, sections were prepared with 2 - 3 μm thickness and serial sections were taken every 100 μm to complete about 20 cut levels across the whole ovarian tissue. For the counting of primordial and growing follicles, HE-stained slides were prepared from all cut levels. Counting was performed on slides digitalized with a Hamamatsu NanoZoomer 2.0 slide scanner using the Hamamatsu viewing software (NDP.view).


Pathological examinations of cohort 1B animals:
Necropsy:
All cohort 1B animals weresacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals will be necropsied and assessed by gross pathology, special attention being given to the reproductive organs.
Organ weights:
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (final body weight), Adrenal glands (fixed), Cauda epididymis, Epididymides, Liver, Ovaries, Pituitary gland (fixed), Prostate (ventral and dorsolateral part together, fixed), Testes, Seminal vesicles including coagulating gland (fixed), Uterus (with cervix).
All paired organs will be weighed together (left and right).
Organ/Tissue fixation:
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Adrenal glands, Cervix uteri, Coagulating glands, Epididymides (fixed in modified Davidson´s solution), Liver, Ovaries (fixed in modified Davidson´s solution), Pituitary gland, Prostate, Seminal vesicles including coagulating glands, Testes (fixed in modified Davidson´s solution), Uterus, Vagina.
The testes, epididymides and ovaries of animals that die or will have to be sacrificed intercurrently will be fixed in 4% neutral-buffered formaldehyde solution.
Histopathology
Histotechnical processing and examination by light microscopy was not performed For technical reasons, the ovaries of all cohort 1B females of all test groups were embedded in paraplast.


Pathological examinations of surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts):
Necropsy: All surplus F1 generation pups that were not used for the following organ weight determinations will be sacrificed under isoflurane anesthesia with CO2. The selected pups for organ weight determination will be sacrificed by decapitation under isoflurane anesthesia. All animals will be necropsied and assessed by gross pathology with special emphasis on the reproductive organs.
Organ weights: The following weights will be determined in up to 10 animals per sex per group sacrificed on schedule: anesthetized animals (final body weight), Brain, Spleen, Thymus (fixed)
Organ/ Tissue fixation: The following organs or tissues of up to 10 animals per sex per group will be fixed in 4% neutral-buffered formaldehyde solution: All gross lesions, Brain, Mammary gland (male and female), Spleen, Thymus, Thyroid glands.
Statistics:
DUNNETT test (two-sided): Food consumption (parental and rearing animals), body weight and body weight change (parental DUN NETT test (two-sided) animals, rearing animals and pups); for the pup
weights, the litter means were used), gestation days, duration of sexual maturation (days to vaginal
opening, days to preputial separation), anogenital distance, anoqenital index
FISHER'S EXACT test (one-sided): Male and female mating indices, male and female FISHER'S EXACT test (one-sided) fertility indices, gestation index, females mated, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups
WILCOXON test (one-sided+) with BONFERRONHOLM: Mating days until day O pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development
WILCOXON test (one-sided-) with BONFERRONHOLM: Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, lactation index
WILCOXON test (two-sided): % live male day x, %live female day x
KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided): Number of cycles and Cycle Length
KRUSKAL-WALLIS and WILCOXON test: Clinical pathology parameters
KRUSKAL-WALLIS and WILCOXON test: Weight of the anesthetized animals and absolute and relative organ weights
WI LCOXON test ( one-sided -): DOFC
KRUSKAL-WALLIS and WILCOXON test: Weight of the anesthetized animals and absolute and relative organ weights
KRUSKAL-WALLIS-H and WILCOXON: Weight of the anesthetized animals and absolute and relative organ weights
Reproductive indices:
Male reproduction data

The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.

Male mating index (%) = number of males with confirmed mating* / number of males placed with females x 100
* defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = number of males proving their fertility* / number of males placed with females x 100
* defined by a female with implants in utero



Female reproduction and delivery data

The pairing partners, the number of mating days until vaginal sperm were detected, and gestational status were recorded for F0 females.
For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:

Female mating index (%) = number of females mated* / number of females placed with males x 100
* defined as the number of females with vaginal sperm or with implants in utero

Female fertility index (%) = number of females pregnant* / number of females mated** x 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with im/plants in utero

Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant* x 100
* defined as the number of females with implants in utero


The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters according to the following formula:

Live birth index (%) = number of liveborn pups at birth / total number of pups born x 100

The implantations were counted1 and the postimplantation loss (in %) was calculated according the following formula:

Postimplantation loss (%) = number of implantations – number of pups delivered / number of implantations x 100
Offspring viability indices:
Viability index (%) = number of live pups on day 4* after birth / number of live pups on the day of birth x 100
* before standardization of litters (i.e. before culling)

Lactation index (%) = number of live pups on day 21 after birth / number of live pups on day 4* after birth x 100
* after standardization of litters (i.e. after culling)
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical observations for males and females:
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the groups.

Clinical observations for females during gestation of F1 litters:
There were no test substance-related clinical findings noted in any female of all dose groups during the gestation period for F1 litter.
One low-dose female (No. 139) had blood in bedding on GD 22.

Clinical observations for females and offspring during lactation of F1 litters:
There were no test substance-related clinical findings noted in all F0 females of all dose groups during the lactation period.
One high-dose female (189) had a complete litter loss and in one mid-dose female (169) all pups were stillborn.

Detailed clinical observations:
Male and female animals of all dose groups (1500, 5000 and 12500 ppm) did not show any abnormalities.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of low-dose F0 males were comparable to the concurrent control values throughout the study. Mean body weight of mid-dose F0 male animals was significantly decreased on study day 105 by -6%. In male animals of the high dose the mean body weights were significantly below the concurrent control values from study day 21 onwards with a maximum by -9% on study day 91.

Mean body weights of the low-and mid-dose F0 females were comparable to the concurrent control values throughout the entire study. In F0 females of the high dose the mean body weight was significantly decreased on GD 20 by -6%, while the mean body weight during premating and lactation were comparable to the concurrent control group.

In comparison to concurrent control, statistically significant decreases and increases of body weight change were observed in all treated F0 male animals as specified below:

• Low-dose males: decrease on study days 42-49 and 98-105, increase study days 91- 98 and 105-112
• Mid dose males: decrease on study days 0-7, 42-49, 77-84, 98-105, increase on study days 70-77, 91-98 and 105-112
• High-dose males: decrease on study days 0-7, 14-21, 28-35, 42-49, 49-56 and 98-105, increase on study days 91-98

Overall, body weight change from study day 0 to 112 was below concurrent control in all treatment groups, however, the decrease gained statistical significance only in the mid-dose males (-7%) and in the high-dose males (-11%).

Body weight change in females of low- and mid-dose group as well as body weight change during premating and lactation in females of high dose group was comparable to the concurrent control group during the entire study.

Body weight change in females of the high-dose group was significantly below to the concurrent control group on study days GD 0-7 (-17%), GD 14-20 (-16%) and GD 0-20 (-14%) during the gestation period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the high-dose F0 males was statistically significantly below the concurrent control values on study days 14, 49, 56, 63 and 105 (maximum by -15% on study day 49) as well as of the mid-dose males on study days 49 (-13%) and 56 (-11%).

Food consumption of the low-, mid- and high-dose F0 females during premating period was comparable to the concurrent control.

Food consumption of the high-dose F0 females was statistically significantly below the concurrent control on GD 7 (-11%) and on GD 20 (-10%) as well as on PND 4 (-16%), PND 10 (-13%) and on PND 18 (-13%).

Food consumption of the low- and mid-dose F0 females during gestation and lactation period was comparable to the concurrent control values.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
At the end of the administration period in F0 females of test group 03 (12500 ppm) absolute large unstained cell (LUC) counts were significantly higher compared to controls. The values were within the historical control range (F0 females, LUC 0.00-0.02 Giga/L). Therefore, this change was regarded as incidental and not treatment related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related, adverse changes among clinical chemistry parameters were observed.
At the end of the administration period, in F0 males of test group 03 (12500 ppm) alanine aminotransferase (ALT) activities were significantly decreased. However, this decrease was below 50% and therefore, this change was most probably due to a liver enzyme induction (PSD Guidance Document, 2007). This was regarded as treatment related but adaptive and not adverse.
In F0 females of test group 03 sodium levels were significantly lower compared to controls, but the values were within the historical control range (F0 females, sodium, 139.7-143.0 mmol/L).
Endocrine findings:
no effects observed
Description (incidence and severity):
Thyroid hormones
In F0 males and females no treatment related effect on the T4 and TSH values could be observed.

In F0 males T4 values in test groups 01, 02 and 03 (1500, 5000 and 12500 ppm) were significantly higher compared to controls. The change was not dose dependent, and all mean values were within the historical control range (F0 males, T4 40.44-72.39 nmol/L). TSH values in males of test groups 01, 02 and 03 were lower compared to study controls, but not statistically significantly. Again, all TSH values were within the historical control range (F0, TSH 3.49-10.31 μg/L). TSH mean value in the study controls was at the upper border of the historical control range. Therefore, the alterations of T4 and TSH in F0 males of test groups 01, 02 and 03 were regarded as incidental and not treatment related.
In F0 dams of test group 03 (12500 ppm) T4 values were significantly higher compared to controls, but the values were within the historical control range. The same was true for the TSH values among these individuals (F0 dams, T4 24.28-46.41 nmol/L; TSH 3.23-6.30 μg/L). Therefore, the T4 change in females of test group 03 was regarded as incidental and not treatment related.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related, adverse changes among urinalysis parameters were observed.
At the end of the administration period, in F0 males of test group 03 (12500 ppm) the incidence of transitional epithelial cells and that of granulated and epithelial casts were significantly in-
creased. These findings were observed only in males, and they were most probably due to an α2u-Globulinuria which was regarded as a species-specific finding without human relevance (Hard, 2018).
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in the glandular stomach of females and the kidneys of males.
The increased incidence of erosion/ulcer in the glandular stomach females of test group 03 (12500 ppm) correlated to the macroscopically detected foci in the glandular stomach and was regarded as treatment related.
In the kidneys of males of test group 03 (12500 ppm) a minimal to slight increase in basophilic tubules was detected. In most animals the kidneys were affected unilaterally. The increase in basophilic tubules was regarded as potentially treatment related but not adverse. An immunohistochemial stain for a2μ in the kidneys was performed with a negative result exemplarily in 2 control and 2 high dose male animals of F0 animals (animal no. 2, 13, 77, 92).
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Fertility:
The female animals (Nos. 103, 105, 108, 112, 134, 138, 148, 149, 172, 182, 184 and 197) which were not pregnant as well as their male mating partners (Nos. 3, 5, 8, 12, 34, 38, 48, 49, 72, 82, 84 and 97), did not show relevant histopathological findings consistent with impaired fertility.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 3 weeks prior to mating to produce the F1 litter,
revealed regular cycles in the females of all test groups including the control. The mean estrous
cycle duration was similar: 4.1 / 3.9 / 4.1 and 4.0 days in test groups 00 - 03, respectively.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Spermanalysis:
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis of F0 males no treatment-related effects were observed.
Reproductive performance:
no effects observed
Description (incidence and severity):
Male reproduction data:
For nearly all F0 parental males, which were placed with females to generate F1 pups,
copulation was confirmed. Male animal No. 12 of control group, which was paired with female animal No. 112 of control group, did not generate a positive vaginal smear Thus, the male mating index was 100% in all treated test groups (01 - 03) and 96% in control group.
Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.
The following male animals did not generate F1 pups or implants:
- Animal Nos. 3, 5, 8 and 12 of control group
- Animal Nos. 34, 38, 48 and 49 of low dose group
- Animal No. 72 of mid dose group
- Animal Nos. 82, 84 and 97 of high dose group
Thus, the male fertility index ranged between 84% and 96%, reflecting the normal range of biological variation inherent in the strain of rats used for this study.

Female reproduction and delivery data:
The female mating index calculated after the mating period for F1 litter ranged between 96% and 100% in all test groups, because no vaginal sperm or implants in utero was detected in female animal No. 112 of the control group.
The mean duration until sperm was detected (GD 0) varied between 1.9 and 2.9 days without any relation to dose.
All impregnated female rats delivered pups or had implants in utero with the following excep-
tions:
• Control group
female No. 103 (mated with male No. 3), female No. 105 (mated with male No. 5) and female No. 108 (mated with male No. 8) did not become pregnant
• Low-dose group
female No. 134 (mated with male No. 34), female No. 138 (mated with male No. 38), female No. 148 (mated with male No. 48) and female No. 149 (mated with male No. 49) did not
become pregnant
• Mid-dose group
female No. 172 (mated with male No. 72) did not become pregnant
• High-dose group
female No. 182 (mated with male No. 82), female No. 184 (mated with male No. 84) and female No. 197 (mated with male No. 97) did not become pregnant
The fertility index ranged between 84% and 96% without any relation to dose.
The gestation index was 100% in control and high-dose group, 95.2% in the low-dose group and 95.8% in the mid-dose group.
The mean duration of gestation ranged between 22.3 days and 22.4 days without any relation to dose.
The mean number of implantation sites was statistically significantly below the concurrent control values in the high-dose group (13.2 / 12.0 / 12.9 and 11.9* implants/dam in test groups 00 - 03, respectively), however, the high-dose value was well within the range of the historical
control data (HCD = 11.1 - 13.9 implants/dam).
There were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any statistically significant differences between the groups (8.2% / 12.3% / 9.3% and 12.9% in test groups 00 - 03, respectively), all values being well within the historical range of the test facility.

Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
12 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: hightest dose tested
Remarks on result:
other: 12500 ppm: about 998 mg/kg bw/d in males, 1136 mg/kg bw/d in females
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: local toxicity in the gastrointestinal tract
Remarks on result:
other: 5000 ppm: about 357 mg/kg bw/d in males, 453 mg/kg bw/d in females
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related adverse clinical signs were observed in any of the F1 generation pups of the different test groups.

F1 rearing animals, Cohort 1 A:
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
One low-dose male animal (No. 432) showed a swollen eyelid (right side) during study days 35 - 48 and one mid-dose male animal (No. 442) had an injury (left shoulder region) on study days 14 - 41. These observations were considered not to be associated with the test compound.

Detailed clinical observations (DCO):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
One low-dose male animal (No. 432) showed a swollen eyelid (right side) on study days 35 – 48, which was also seen on the respective days of detailed clinical observation, i.e. study days 35 and 42. Further, one mid-dose male animal (No. 442) had an injury (left shoulder region) from study day 14 - 41. This finding was also seen on the respective days of detailed clinical observation, i.e. study days 14, 21, 28 and 35.
These observations were considered not to be associated with the test compound.

F1 rearing animals, Cohort 1B:
Clinical observations for males and females:
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F1B animals in any of the groups.
One control male animal (No. 212) showed an anomaly of teeth from study day 7 onwards and one control male animal (No. 213) showed an injury in the left shoulder region from study day 49 onwards. An anomaly of teeth was also seen in one mid-dose female animal (No. 352) from study day 7 to 41. Further, an injury at the right eye was observed in one high-dose female animal (No. 381) from study day 52 onwards. These observations were considered not to be associated with the test compound.

Detailed clinical observations (DCO)
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
One control male animal (No. 212) showed an anomaly of the teeth from DCO day 7 onwards and one control male animal (No. 213) showed an injury in the left shoulder region from DCO day 49 onwards. An anomaly of the teeth was also seen in one mid-dose female animal (No. 352) from study day 7 to 41. This finding was also seen on the respective days of detailed clinical observation, i.e. study days 7, 14, 21, 28 and 35. These observations were considered not to be associated with the test compound.
Mortality / viability:
no mortality observed
Description (incidence and severity):
F1 rearing animals, Cohort 1 A:
There were no test substance-related or spontaneous mortalities in any of the groups.

F1 rearing animals, Cohort 1B:
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F1 pup body weights did not show significant differences between control and all treatment groups. However, mean pup weights were slightly lower in the high-dose group than in the control group (by about 5%) at the end of lactation, though the difference did not become statistically significant. This minor change reflects the decrease in high-dose pup body weight gain towards the end of lactation.
Likewise, there was no significant difference in mean pup body weight change noted between control and test groups 01 and 02. Mean pup body weight change of the high-dose animals was also comparable to the control group throughout the first two weeks of lactation but was statistically significantly below the concurrent control during the last week of lactation (PND 14 – 21: -13%, -11% and -12% in males, females and both sexes combined, respectively).

F1 rearing animals, Cohort 1 A:
Mean body weights of the high-dose male animals were decreased throughout the study with a maximum by -8.6% on study day 0, the difference to the control gained statistical significance from study day 0 to 35 and on study 56. In high dose female animals the mean body weight was significantly decreased on study day 7 (-6.5%). High-dose female body weight remained below control throughout the study, however, the difference to control was rather small and gained no statistical significance.
Mean body weights of the low-and mid-dose males and females were comparable to the concurrent control throughout the study.
Body weight change of all test substance-treated male and female animals was comparable to the concurrent control values throughout the study.
The statistically significantly decreased body weight change in the mid-dose males during study days 49 - 56 was considered as spontaneous in nature.

F1 rearing animals, Cohort 1B:
Generally, mean body weights of the mid- and high-dose F1B male animals were below the concurrent control throughout the study, the difference amounted to 6 – 7% below control across both groups. The decreases gained statistical significance on study days 28, 42 and 49 in the high-dose group as well as on study days 14, 42 and 49 in the mid-dose group.
In female F1B animals mean body weights of all treatment groups were largely comparable to the control. A statistically significant difference to the control was only noted for the mid-dose group on study day 49 (-6%).
Mean body weights of the low-dose F1B animals were comparable to the concurrent control throughout the study.
Body weight change of the high-dose F1B males was statistically significantly below the con-
current control values during study days 21 - 28, 35 – 42 and 0 - 49 (up to 25%) and of the mid-dose males during study days 7 - 14, 21 - 28, 35 - 42, 42 - 49 and 0 - 49 (up to 19%).
Further, in female animals of the mid-dose body weight change was also statistically significantly below the concurrent control value on study days 35 - 42, and 0 - 49 (up to 21%)
Body weight change of the low-dose F1B males as well as low- and high-dose females were comparable to the concurrent control values throughout the entire study.

The effects on the body weight change are expected to be secondary to the local toxicity in the gastrointestinal tract based on the irritant properties of the test substance.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
F1 rearing animals, Cohort 1 A:
Food consumption of all test substance treated male and female rats was comparable to the concurrent control throughout the study.
Food consumption of low-dose male animals was significantly increased on study day 56 and in low- and mid-dose females significantly decreased on study day 49. These changes were rather inconsistent and, thus, considered not to be treatment-related.

The intake of 2-Ethylhexyl acrylate (in mg/kg bw/d) was calculated on the basis of most recent individual body weights in each test group.
1500 ppm: males 129.0 mg/kg bw/d, females 139.8 mg/kg bw/d
5000 ppm: males 431.7 mg/kg bw/d, females 468.2 mg/kg bw/d
12500 ppm: males 1093.2 mg/kg bw/d, females 1171.9 mg/kg bw/d

F1 rearing animals, Cohort 1B:
Food consumption of all test substance treated animals was comparable to the concurrent
control throughout the study.
There was a number of sporadic statistically significant food consumption decreases, such as
in male animals of mid- and high-dose group on study day 28, in female animals of low-dose group on study days 14 and 42 as well as in female animals of the mid-dose group on study days 42 and 49. None of these changes were considered to be treatment-related.

The intake of 2-Ethylhexyl acrylate (in mg/kg bw/d) was calculated on the basis of most recent individual body weights in each test group.
1500 ppm: males 131.3 mg/kg bw/d, females 137.8 mg/kg bw/d
5000 ppm: males 445.1 mg/kg bw/d, females 461.5 mg/kg bw/d
12500 ppm: males 1106.7 mg/kg bw/d, females 1176.0 mg/kg bw/d
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1 generation (cohort F1A)
No treatment-related changes among hematological parameters were observed.
At the end of the administration period in F1A females of test group 11 (1500 ppm) prothrombin time (Hepatoquick’s test, HQT) was significantly shortened. In females of test group 12 (5000 ppm) relative monocyte counts were significantly lower compared to controls. However, both alterations were not dose dependent and therefore, they were regarded as incidental and not treatment related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1 generation (cohort F1A):
No treatment-related, adverse changes among clinical chemistry parameters were observed.
At the end of the administration period in females of test group 13 (12500 ppm) alkaline phosphatase (ALP) activities and glucose levels were significantly increased whereas creatinine levels were significantly decreased. Creatinine values were within the historical control range and glucose levels were marginally above their range (F1A females, creatinine 26.3-35.4 μmol/L, glucose 5.01-6.26 mmol/L). Therefore, creatinine decrease was regarded as incidental
and not treatment related. The isolated glucose increase was assessed as non-adverse if at all treatment related (ECETOC Technical Report No. 85, 2002).
Alkaline phosphatase activity mean value was 33.1% higher compared to the study control mean, and it was above the historical control values (F1A females, ALP 0.63-1.05 μkat/L). However, because no other liver parameter was relevantly changed, liver weights were not altered, and no histologic finding in the liver was observed a liver cell affection can be excluded. Therefore, the slight ALP increase was regarded as maybe treatment related, but non-adverse (ECETOC Technical Report No. 85, 2002).
In F1A females of test group 11 (1500 ppm) glucose levels were significantly increased, but the change was not dose dependent. Therefore, this alteration was regarded as incidental and not treatment related.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1 generation (cohort F1A):
No treatment-related, adverse changes among urinalysis parameters were observed.
At the end of the administration period, in F1A males of test group 13 (12500 ppm) the incidence of transitional epithelial cells and that of granulated and epithelial casts were significantly increased. Significantly increased incidences of casts were also observed in males of test group 12 (5000 ppm). These findings were observed only in males, and they were most probably due to an α2u-Globulinuria which was regarded as a species-specific finding without human relevance (Hard, 2018).
Sexual maturation:
no effects observed
Description (incidence and severity):
F1 generation pups/litters:
The sex distribution and sex ratios of live F1 pups did not show substantial differences between the control and the test substance-treated groups, slight differences were regarded to be spontaneous in nature. The apparently higher rate of male F1 pups and correspondingly lower rate of female F1 pups in test group 01 was assessed as spontaneous in nature, because no dose response occurred and the distribution ranged within the historical control data (males: 43.3% – 55.5%, females: 44.5% - 56.7%).

Vaginal opening:
Each female F1 pup, which was selected to become a rearing female, was evaluated for commencement of sexual maturity. The first day when vaginal opening was observed was PND 31, the last was PND 43. The mean number of days to reach the criterion in the control and 1500, 5000 and 12500 ppm test groups was 35.4; 35.3; 35.8 and 36.9** (** = p≤0.01) days, respectively. The mean body weight on the day, when vaginal opening was recorded, amounted to 115.2 g, 114.6 g, 114.7 g and 118.2 g in test groups 00-03.
A statistically significant delay in vaginal opening of about one day beyond the concurrent control was observed in the female F1 offspring of the high-dose group (12500 ppm). The delay is within the historical control range of the test facility. There was a mild effect on post-weaning body weight development in the affected females around the timing of puberty (around study day 7 in the F1A cohort) notable. On the other hand, there was no effect on estrous cyclicity or the integrity of sexual organs in the investigated F1 females, including differential ovarian follicle count, during later life. Thus, there is evidence that this finding is due to an unspecific minimal delay of general development rather than a specific effect on the timing of puberty. Overall, it is regarded as of no toxicological relevance.

Preputial separation:
Each male F1 pup, which was selected to become a rearing male, was evaluated for com-
mencement of sexual maturity. The first day when preputial separation was observed was PND 39, the last was PND 55. The mean number of days to reach the criterion in the control and 1500, 5000 and 12500 ppm test groups was 44.0, 44.3, 44.4 and 44.8 days, respectively. The mean body weight on the day, when preputial separation was recorded, amounted to 188.3 g, 190.2 g, 185.9 g and 185.0 g in test groups 00-03.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distance and anogenital index of all test substance treated male and female pups was comparable to the concurrent control.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The percentage of male pups having nipples/areolae was not influenced by the test substance when examined on PND 13 and during re-examination on PND 20.
Description (incidence and severity):
F1 rearing animals, cohort 1A

Absolute organ weights
When compared the control group 10 (set to 100%) with test groups 11, 12 and 13, the mean absolute weights of following organs were significantly decreased: terminal body weight, adrenal glands, heart and spleen.
All other mean absolute weight parameters did not show significant differences.

Relative organ weights:
When compared the control group 10 (set to 100%) with test groups 11, 12 and 13, the mean relative weights of kidneys and liver in the high dose males were significantly increased.
All other mean relative weight parameters did not show significant differences.
The terminal body weight of males of test group 13 (12500 ppm) was significantly decreased (304.83g; - 7.1%). The significant absolute weight decreases of adrenal glands and spleen in males of test group 13, as well as the significant decreases in heart weights in males of test group 13 and females of test group 12 and 13 were not accompanied by significant decreases of the corresponding relative weights. No histopathological correlate could be found and therefore the weight decreases were regarded as secondary to the decrease in terminal body weight.
The significant decrease of absolute spleen weights in females of test group 12 did not show
dose dependency and was regarded as incidental. The significantly increased relative liver weights in males (2.607%, +6%) was within the historical control range (2.552% - 2.716%). No histopathological correlate could be detected and therefore the finding was regarded as incidental and not treatment related.
The increased relative kidneys weight in males of test group 13 (0.738%, +16.5%) was above the historical control range (0.624 – 0.663%) but the mean absolute weight kidney weight was not significantly increased. The change was regarded as secondary to the reduced body weight.

F1 rearing animals, cohort 1B:

Absolute organ weights:
When compared test groups 11, 12, and 13 with the control group 10 (set to 100%), the mean absolute weights of following organs were significantly increased: terminal body weight, adreanal glands, epididymides, prostate, uterus,
All other mean absolute weight parameters did not show significant differences.

Relative organ weights:
When compared test groups 11, 12, and 13 to the control group 10 (set to 100%), the mean relative weights of following organs were significantly increased. Liver, pituitary gland, testes.
All other mean relative weight parameters did not show significant differences.
The terminal body weight of males of test group 12 and 13 was significantly decreased (306.97g, -6.1%; 303.76g, -7.1%). The absolute weights of the adrenal glands (60 mg, -11%), the epididymides (0.887, -4.4%) and the prostate (0.668g, -8.5%) in males of test group 13 were significantly decreased. The corresponding mean relative weights were not significantly changed. The relative weight decreases were regarded as most likely secondary to the decreased terminal body weight.
The significantly decreased mean absolute uterus weight in females of test group 12 did not display dose dependency and was regarded as incidental and not treatment related.
The significant increase of relative liver weights of males of test group 11, 12 and 13 (2.620%, +9.6%; 2.696%, +12.8%; 2.734%, +14.4%) and females of test group 13 (2.656%, +4.1%), of the pituitary gland of males of test group 12 and 13 (0.003%, +12.6%; 0.003% +20.1%) and of testes of males of test group 12 and 13 (1.133%, +5.8%; 1.141% +6.5%) was not accompanied by changes in absolute weights and was regarded as secondary to the reduced body weight.

Surplus F1 generation pups on PND 22:
Absolute organ weights:
All mean absolute weight parameters showed no significant differences.
Relative organ weights:
When compared test groups 01, 02, and 03 with the control group 00 (set to 100%), the mean absolute weights of following organs were significantly increased.
The decreased brain weight in males of test group 13 (3.021%, -5.7%) was regarded as secondarily to the decreased terminal body weight (51.53g, -5.1%, not significant).
All other mean relative weight parameters showed no significant differences.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related, adverse findings were observed.
A few F1 pups showed spontaneous findings at gross necropsy, such as postmortem autolysis.
These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were considered not to be associated with the test substance.

F1 rearing animals, cohort 1A:
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

F1 rearing animals, cohort 1B:
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Surplus F1 generation pups on PND 22:
No treatment-related gross changes were observed.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 rearing animals, cohort 1A:
Treatment-related findings were observed in the glandular stomach of females and the kidneys of males.
The increased incidence of erosion/ulcer in the glandular stomach females of test group 13 (12500 ppm) is regarded as treatment related and locally adverse. In the kidneys of males of test group 11, 12 and 13 an increase in basophilic tubules were detected. The kidneys were only minimally to mildly and mostly unilaterally affected. The increase in basophilic tubules was regarded as potentially treatment related but not adverse. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

F1 rearing animals, cohort 1B:
Histopathological examination was not performed.

Surplus F1 generation pups on PND 22:
Histopathological examination was not performed.

Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
There was no evidence that the test substance produced any developmental immunotoxicity. Absolute and relative lymphocyte subpopulation cell counts in the spleen tissue (B-, Tlymphocytes, CD4-, CD8-T-lymphocytes and natural killer (NK) cells) displayed no treatment-related changes.
F1 generation pups/litters:
Litter data
Pup number and status at delivery:
As a consequence of the lower number of implants the mean number of live F1 pups delivered per dam (average live litter size) was statistically significantly below the concurrent control value in the high-dose group (12.0 / 10.8 / 11.2 and 10.3** pups/dam, respectively in test groups 00 - 03), but inside the historical control range (HCD = 10.3 - 12.7 pups/dam).
The rate of liveborn pups indicated by live birth indices was 98.4% / 95.2% / 97.8% and 99.6% in test groups 00 – 03, respectively, showing no significant difference between the groups. The number of stillborn pups was not significantly different between the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study. The slightly higher numbers in test groups 01 and 02 were due to two individual litters with a high number of stillborn pups (No. 141, 8/12 stillborn) or a total litter loss (No. 169, 6/6 stillborn).
Pup viability/mortality:
The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 100% / 97.8% / 99.4% and 93.5% in test groups 00 - 03 without showing significant differences between the groups.
The lactation index indicating pup survival on PND 4 - 21 was 99.5% / 100% / 100% and 100%
in test groups 00 - 03 without showing any association to the treatment.
The respective values reflect the normal range of biological variation inherent in the strain used in this study.

F1 rearing animals, Cohort 1A:
Estrous cycle data, generated during 2 weeks, revealed regular cycles in the females of all test
groups including the control. The mean estrous cycle duration was comparable between the groups: 4.3 / 4.1 / 3.9 and 4.1 days in test groups 10 - 13, respectively.
Differential ovarian follicle count:
The results of the differential ovarian follicle count (DOFC) – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – did not reveal significant differences between the control group 10 and animals of test group 13:

F1 rearing animals, Cohort 1B:
Estrous cycle data, generated during 2 weeks, revealed regular cycles in the females of all test
groups including the control. The mean estrous cycle duration was comparable between the groups: 4.1 / 4.0 / 4.2 and 4.3 days in test groups 10 - 13, respectively.

Thyroid hormones (surplus pups PND4 and PND22, cohort F1A);
Neither in F1 PND4 as well as PND22 pups nor in F1A males and females any treatment related, adverse effect on the T4 and TSH values could be observed.
In female PND4 pups of test group 01 (1500 ppm) T4 values were significantly increased, but the change was not dose dependent. Therefore, this alteration was regarded as incidental and not treatment related.
In males PND22 pups of test group 03 (12500 ppm) T4 values were significantly increased. However, the values were within the historical control range (male PND22 pups, T4 42.79-71.39 nmol/L). TSH values were lower compared to study controls, but not statistically significantly. The TSH mean values was slightly below the historical control range (males, PND22 pups, TSH 2.46-4.82 μg/L). In female PND22 pups no changes of T4 and TSH values could be observed. Therefore, also in male PND22 pups of test group 03, the alterations were regarded as incidental and not treatment related.
In F1A females of test groups 12 and 13 (5000 and 12500 ppm) T4 values were significantly
higher compared to controls and the values were slightly above the historical control range. TSH values among these individuals were within the historical control range (F1A females, T4 26.66-47.73 nmol/L; TSH 2.95-4.98 μg/L). However, no thyroid weight changes and no histopathologic findings in the thyroids were observed. Therefore, the isolated slightly higher T4 value in F1A females of test group 13 was regarded as maybe treatment related, but nonadverse (ECETOC Technical Report No. 5, 2002).

Lymphocyte subpopulations in spleen (cohort F1A):
No alterations in the absolute and relative lymphocyte subpopulation cell counts in the spleen tissue (B-, T-lymphocytes, CD4-, CD8-T-lymphocytes and natural killer (NK) cells) were observed in the F1A generation at PND90 in both sexes.

Spermanalysis (cohort F1A):
Concerning the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididiymidis, no treatment-related effects were observed.
Regarding motility of the sperms in males of test group 13 (12500 ppm) this parameter was significantly decreased, and the mean value was below the historical control range (motility 79-92 %). Among the individuals in this group there was one animal (no. 466) with severe histologic findings in the testes which was regarded as a spontaneous rather than a compound related effect. When eliminating the results of this animal from the statistics of the spermanalysis, motility was still weakly significantly lower compared to the study control but mean as well as median value of test group 13 was within the historical control range. No other sperm parameter was altered in this test group and no compound-related, histologic finding in the male reproductive organ was observed. Therefore, reduced motility in F1A males of test group 13 were regarded as non-adverse if at all treatment related.
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
12 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: 12500 ppm: about 998 mg/kg bw/d in males, 1136 mg/kg bw/d in females
Dose descriptor:
NOAEL
Remarks:
developmental immunotoxicity
Generation:
F1
Effect level:
12 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: 12500 ppm: about 998 mg/kg bw/d in males, 1136 mg/kg bw/d in females
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity
Generation:
F1
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: local toxicity in the gastrointestinal tract
Remarks on result:
other: 5000 ppm: about 357 mg/kg bw/d in males, 453 mg/kg bw/d in females

Table 1 Intake of the test substance (F0 generation parental animals):

 

Test group 01 (1500 ppm)

Test group 02 (5000 ppm) Test group 03 (12500 ppm)
F0 males 96.4 313.2 794.3
F0 females (premating) 124.8 415.8 1048.7

F0 females

-lactation period

-gestation period

 

101.6

146.9

 

340.3

495.6

 

825.2

1192.4

Table 2 Intake of the test substance (F1 rearing animals, Cohort 1A):

  Test group 11 (1500 ppm) Test group 12 (5000 ppm) Test group 13 (12500 ppm)
males 129.0 431.7 1093.2
females 139.8 468.2 1171.9

Table 3 Intake of the test substance (F1 rearing animals, Cohort 1B):

  Test group 11 (1500 ppm) Test group 12 (5000 ppm) Test group 13 (12500 ppm)
males 131.3 445.1 1106.7
females 137.8 461.5 1176.0
Conclusions:
Thus, under the conditions of the present extended one-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 5000 ppm (about 357 mg/kg bw/d in males, 453 mg/kg bw/d in females) in the F0 parental and the F1 adolescent/adult rats, based on evidence for local toxicity in the gastrointestinal tract which conveyed to systemic toxicity such as decreased body weight/body weight gain across generations and cohorts, at the LOAEL (Lowest Observed Adverse Effect Level) of 12500 ppm (about 998 mg/kg bw/d in males, 1136 mg/kg bw/d in females).

The NOAEL for fertility and reproductive performance is 12500 ppm (about 998 mg/kg bw/d in males, 1136 mg/kg bw/d in females), the highest dose tested.

The NOAEL for developmental toxicity in the F1 progeny is 12500 ppm (about 998 mg/kg bw/d in males, 1136 mg/kg bw/d in females), the highest dose tested.

The NOAEL for developmental immunotoxicity for the F1 progeny is 12500 ppm (about 998 mg/kg bw/d in males, 1136 mg/kg bw/d in females), the highest dose tested.
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From May 19, 1992 to April 19, 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Wistar rats (Chbb = THOM (SPF))
- Source: Karl THOMAE, Biberach an der Riss, Germany
- Age at study initiation: (P): 35 ± 1 days
- Weight at study initiation: (P) Males: 140.0 (127 - 154) g; Females: 118.8 (106 - 130) g
- Housing: Single in type DK III stainless steel wire mesh cages, with the following exceptions: during mating periods, the males designated for mating were kept individually in Makrolon cages, type M III; for the overnight mating the females were put into the cages of the males. From day 18 of pregnancy until day 14 after birth, the pregnant animals and their litters were also housed in Makrolon type M III cages.
- Diet (ad libitum): Kliba maintenance diet rat/ mouse/hamster GLP 343 meal (KLINGENTALMUEHLE AG, Kaiseraugst, Switzerland)
- Water (ad libitum): Tap water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: drinking water
Vehicle:
water
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: a maximum of 3 weeks.
- Proof of pregnancy: sperm in vaginal smear referred as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The content of Acrylic acid in the aqueous solutions was determined by gas chromatography.
Duration of treatment / exposure:
Exposure period: F0: at least 70 days before the mating and afterwards during the gestation and lactation periods.
F1: at least 98 days before the mating and afterwards during the gestation and lactation periods.
Premating exposure period (males): at least 70 days
Premating exposure period (females): at least 70 days
Duration of test: approx. 12 months
Frequency of treatment:
continuously
Remarks:
Doses / Concentrations:
0, 500, 2500 and 5000 ppm (corresponding to approx. 0, 53, 240 and 460 mg/kg bw/day)
Basis:
nominal in water
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Positive control:
none
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS:
All parental animals were checked daily for clinically evident signs of toxicity. Particular attention was given to the nesting, littering and lactation behaviour of the dams, but only special findings were documented.

BODY WEIGHT:
- Parental animals: generally, body weight was determined once weekly until the end of the study, and at the time of necropsy.
- F0 and F1 fertilized females and females with litter: body weight was determined on the day of sperm evidence in the vaginal smear and thereafter on days 7, 14 and 20 of gestation, one day after parturition, and on days 7, 14 and 21 post-parturition.
- Females without positive evidence of sperms: body weight was not determined during the mating interval.
- Females without litter: body weight was not determined during the lactation phase.

FOOD CONSUMPTION:
- F0 and F1 parental animals: food consumption was determined once weekly (over 7 days) during the period prior mating.
- Pregnant females: food consumption was determined for days 0-7, 7-14, 14-20 post coitum (pc).
- Lactating females: food consumption was determined for days 0-4, 4-7, 7-14 post parturition (pp).
- F0 and F1 dams between day 14 and 21 pp: food consumption was not determined for the F0 and F1 dams between day 14 and 21 pp, since during this period the pups started consumption of solid food; therefore there was no point in such a measurement.
- Females during mating period, females without positive evidence of sperms, females without litter: food consumption was not determined respectively during mating period, gestation period or lactation phase.


WATER CONSUMPTION:
- F0 and F1 parental animals: water consumption was determined once weekly (over 3 days) during the period prior mating.
- Pregnant females: water consumption was determined for days 0-1, 6-7, 13-14, 19-20 post coitum (pc).
- Lactating females: water consumption was determined for days 1-2, 3-4, 6-7, 13-14 post parturition (pp).
- F0 and F1 dams from day 20 and 21 pp: water consumption was not determined for the F0 and F1 dams for days 20 - 21, since during this period the pups started consumption of water; therefore there was no point in such a measurement.
- Females during mating period, females without positive evidence of sperms, females without litter: water consumption was not determined respectively during mating period, gestation period or lactation phase.


INTAKE OF TEST SUBSTANCE:
The intake of test substance (IT, in mg/kg bw/day) was calculated according to the following formula:

ITx = WCx * D / BWy

D = dose in ppm
WCx = daily water consumption on day x; in g
BWy = body weight on day y; in g


Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
The pups (F1 and F2 litters) were examined as soon as on their day of birth for the determination of the total number of pups and the number of liveborn and stillborn pups (pups died on day of birth prior the first examination). Thereafter the pups were checked twice daily on workdays (once a day on week ends and public holidays) for mortality (i.e. dead and moribund pups) and the mortality (number and percentage) was determined for the day of birth (i.e. day 0) and for the periods: days 1 - 4, 5 - 7, 8 - 14 and 15 - 21 of lactation. Pups that died accidentally and had to be sacrificed because of maternal death were not considered for calculation. The number of surviving pups was determined for days 0, 4, 7, 14 and 21 of lactation and served for the calculation of the viability index and the lactation index.

The sex of the pups was determined on day 0 and day 21 (measurement of the anogenital distance, which is known to be greater in male pups than in females), and the sex ratio was calculated according to following formula:

- Sex ratio = number of live male or female pups on day 0/21 * 100 / number of live male and female pups on day 0/21

The pups were weighed on days 1, 4, 7, 14 and 21 after birth, and they were examined daily for clinical symptoms or gross morphological abnormalities. The determination of the relative organ weight was based on the pup body weight on day 21 after birth. The bodies of the sacrificed pups were examined for external abnormalities and the organs also were subjected to gross pathology; skeletal staining according to the modified Dawson´s method and/or further processing of the head according to Wilson´s method was done in case of abnormal findings. Stillborn pups as well as pups that died during weaning also were subjected to necropsy.

Development stages / Behavioral tests:
Physical development was assessed by monitoring pinna unfolding, opening of the auditory canal and opening of the eyes. Additional tests
were performed to assess grip reflex, hearing and pupillary reflex as follows :
- Grip reflex: Tested on day 13 after birth by placing front paws onto 3-mm diameter rod. For a positive response, the animal had to grip the bar and pull itself up.
- Hearing test: On day 21 after birth, animals were placed in a soundproof box and exposed to a sound (0 .1 sec, 5000 Hz, about 90 dB); a startle reflex was considered a response to this stimulus.
- Pupillary reflex: On day 2l after birth, pupillary constriction reflex was assessed by shining a penlight on the eye and observing the reaction.

Postmortem examinations (parental animals):
SACRIFICE
Parental animals were killed by decapitation under CO2 anaesthesia and examined macroscopically.

GROSS NECROPSY
Terminal body weights as well as the weights of liver, kidneys, epididymides and testes were recorded .

HISTOPATHOLOGY / ORGAN WEIGHTS
Liver, kidneys and stomach (non-glandular and glandular).
Postmortem examinations (offspring):
SACRIFICE
Pups were killed by CO2 asphyxiation, examined externally, eviscerated and their organs assessed macroscopically.


GROSS NECROPSY
External and internal examinations including the cervical, thoracic, and abdominal viscera.


HISTOPATHOLOGY
Vagina, cervix, uterus, ovaries, oviducts, testes, epididymides, seminal vesicles, coagulation gland, oesophagus and duodenum.
Statistics:
The statistical assessment of the different data obtained within the present study was based on following methods, depending on the parameters considered: Dunnett test, Fisher`s exact test and Wilcoxon test.
Reproductive indices:
Mating and fertility indices were calculated according to following formulas:

- Male mating index (%) = number of males with confirmed mating * 100 / number of males placed with females

- Male fertility index (%) = number of males proving their fertility * 100 / number of males placed with females

Remark:
Males were defined as “with confirmed mating” by the presence of vaginal sperm in the female, or by the production of a litter, or by the presence of fetuses in the uterus.
Males were defined as “proving their fertility” by female giving birth to a litter or having pups or fetuses in the uterus.

Reevaluation of fertility:
If an animal of the F0 or F1 generation parental animals had not produced any offspring after the scheduled mating of F0 parents (to get F1 litter) or after the scheduled mating of F1 parents (to get of F2 litter), those animals treated with the test substance were mated with fertile animals of the control groups. Animals of the control groups which seemed to be infertile were mated with mating partners with proven fertility of the controls.
After fertility had been reevaluated, the animals were sacrificed and subjected to gross-pathological and histopathological assessments. The uteri of the females reevaluated for fertility were examined for live and dead implantations. In the case of an apparently non-pregnant animal or of an empty uterus horn in the case of single-horn pregnancy, the uterus was stained with sodium sulfide and assessed for early implantations. Then the uteri were rinsed carefully under running water. After these examinations were completed, the uteri were transferred to the pathology lab for further fixation and evaluation.

Offspring viability indices:
- Viability index (%) = number of live pups on day 4 after birth * 100 / number of liveborn pups on the day of birth

- Lactation index (%) = number live pups on day 21 after birth * 100 / number of live pups on day 4 after birth

Remark:
Day 4 after birth preceded standardization of the litters.
Day 21 after birth followed standardization of the litters.
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
**** F0 GENERATION PARENTAL ANIMALS ****

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- There were no mortalities in any of the F0 generation parental animals in any of the groups.
- No clinical signs which might be attributed to the test substance were detected in male or female F0 generation parental animals. The 3 concentrations administered in the drinking water did not lead to disturbances of the general behavior in any of the F0 parental animals.
 - There were no particular substance-related clinical findings in F0 females during the gestation period for F1 litter. Insufficient nesting activity was observed for several dams of all groups including the controls.
 - No substance-related clinical findings were recorded for the F0 dams during the F1 lactation period.

FOOD CONSUMPTION (PARENTAL ANIMALS)
In general, the food consumption of the males (during the premating period) and of the females (during premating, gestation and lactation periods) of all test groups was not influenced by the test substance administration. It was, however, slightly, but statistically significantly reduced in the high dose males during the first week of the premating period and in the females of 5000 ppm test group during the second week of the lactation period.
The sporadic and only marginal reductions in food consumption of the 5000 ppm rats are probably related to the reduced consumption of aqueous acrylic acid solutions of these animals and thus are likely indirectly associated with the administration of the test substance. All other observable differences between the groups are without biological relevance, because they are not dose-related; this includes the statistically significantly increased food consumption of the low dose females during study weeks 0-1 and 6-7 of the premating period.

BODY WEIGHT (PARENTAL ANIMALS)
In the F0 males statistically significant reductions in mean body weights were seen in the highest dose group (5000 ppm) from week 12 until week 20 of the study period. Body weight changes of the high dose males were statistically significantly diminished only at certain study intervals (weeks 0-1, 6-7, 11-12); if calculated for the total study period (weeks 0-20), body weight gain of the high dose males was about 9% lower than that of the respective controls.
Body weights and weight gains of the substance-treated females were similar to control values during the premating period and during gestation and lactation. Only during the first week of gestation did the high dose dams gain statistically significantly less weight than the corresponding controls.
Finally the impairments in body weight/body weight gain in the high dose F0 males and - to a lesser extent - in the F0 females are assessed as being substance-related effects. All other statistically significant differences in body weights and body weight gains are considered unrelated to the test substance because the values were not influenced in a dose-dependent manner and/or are within the biological range of variation.

WATER CONSUMPTION (PARENTAL ANIMALS)
The water consumption of the high dose male and female animals was clearly reduced. This reduction was statistically significant during the premating period. It was also diminished in the 5000 ppm females during gestation and lactation of F1 litter. In total the 5000 ppm males consumed about 11% and the high dose females about 13% less drinking water (aqueous acrylic acid solutions) then the respective controls during the first 10 study weeks. The marked reduction in the drinking water consumption of the high dose rats was associated with the test substance administration. All other observable differences between the groups in respect to water consumption are without biological relevance, because they are not dose-related; this includes the statistically significantly increased water consumption of the 500 ppm female animals during premating weeks 6-7 and 9-10.

MALE REPRODUCTIVE FUNCTION (PARENTAL ANIMALS)
For all F0 males which were placed with females to generate F1 pups mating was confirmed; thus, the male mating index was 100% in all groups. For nearly all F0 males fertility could be confirmed within the scheduled mating interval; the fertility index varied between 92% and 96% with no treatment related effect. Thus, the fertility of the F0 generation parental males was not adversely influenced by the administration of aqueous acrylic acid solutions.

FEMALE REPRODUCTIVE FUNCTION (PARENTAL ANIMALS)
The female mating index calculated after the mating period for F1 litter was 100% for all groups. The mean duration until sperm was detected (day 0 pc) varied between 1.8 and 3.8 days and was statistically significantly longer for the high dose dams; the high dose value (3.8 days), however, is substantially similar to the mean cohabitation time value of the control group (3.2 days) of the second parental generation (F1 animals) and therefore was not considered treatment-related. Only one or two females in all groups, including the controls, did not become pregnant within the scheduled mating interval. The fertility index varied between 92% and 96% without any dose-response relationship. All females in question except the 2 low dose females proved to be fertile after being mated again with control males. The mean duration of gestation was similar in all groups and the gestation index reached 100% for all groups. The mean number of pups delivered/dam was uninfluenced by the test substance administered. The number of liveborn and stillborn pups was comparable between the groups, and the live birth index was 98% in test groups. Thus, the administration of aqueous acrylic acid solutions did not adversely affect reproduction and delivery data of the FO generation parental females.


GROSS AND HISTOPATHOLOGICAL FINDINGS
- Thickening of the limiting ridge (margo plicatus) of the forestomach in most male and female rats.
- Minimal hyperkeratosis at the limiting ridge of the forestomach in most male and all female rats.
- Edema in the submucosa of the glandular stomach of 2 male and 10 female rats, minimal in all cases.

**** F1 GENERATION PARENTAL ANIMALS *****

MORTALITY / CLINICAL OBSERVATIONS
- One male animal of 2500 ppm test group had to be sacrificed in a moribund state due to a severe skin lesion on the base of the tail. There were no other unscheduled mortalities in any of the test groups.
 
- No clinical signs which might have been attributed to the test substance administered were detected in male or female F1 generation parental animals. The 3 doses administered in drinking water did not lead to disturbances of the general behavior in any of the F1 parental animals. One male animal of 2500 ppm test group developed a severe skin lesion on the base of the tail and was sacrificed in a moribund state. Another male of the same dose group showed unilateral chromodacryorrhea. The clinical findings which occurred in just two intermediate dose males were spontaneous in nature.
 
- No particular clinical findings were noted for F1 dams with positive sperm detection except insufficient or no nesting activity, which was recorded for several dams of all groups (0, 500, 2500 and 5000 ppm) and which occurred without a clear dose-response relationship. One female of the low dose group showed vaginal hemorrhage towards or after the end of the gestation period (days 23 - 26 pc), and was not able to deliver the pups, which were palpable earlier in the abdomen of this dam. After day 26 pc, no pups could be palpated for this dam.
 
- There were no substance-related clinical findings in the F1 dams during the lactation of F2 litters. Only one dam of the low dose group and one dam of the high dose group did not nurse their pups properly; all pups of high dose dam were cannibalized and/or died intercurrently. Furthermore, another low dose dam showed blood in bedding during the first days of the lactation period, and was not able to deliver its litter completely. It delivered only 2 pups which were cannibalized on day 1 pp.

FOOD CONSUMPTION
The mean food consumption of the males and females of 5000 ppm test group was clearly reduced during the premating period, the differences in comparison to the controls being statistically significant at most time intervals. In total, the high dose males consumed about 9% and the females about 8% less food than the respective control animals during the premating phase. Food intake was also statistically significantly diminished in the females of this test group (5000 ppm) during the gestation period (days 7-20 pc) and during the lactation period (days 7-14 pp only). The reduction in food consumption of the 5000 ppm males and females was considered to be related to the administration of the test substance. All other differences in food consumption between the groups are without any biological relevance.
 
WATER CONSUMPTION
In comparison to the respective control values the water consumption of the 2500 and 5000 ppm F1 males and females was distinctly lower during the premating period, the differences being statistically significant at all intervals in the high dose level and in several but not all intervals at the intermediate dose. In total a clear dose-response relationship was observed: high dose males consumed about 18%, intermediate dose males about 9% less water than control males; for high dose females about 27% and for intermediate dose females about 13% less water intake than in the female controls was recorded. Water consumption was also reduced during gestation and lactation periods in these test groups, again more pronounced in the high than in the 2500 ppm group. The water consumption of the animals of the low dose group (500 ppm) reached or even exceeded the relevant control values during premating, gestation and the lactation periods. The distinct reductions in the drinking water consumption in both sexes at 2500 and 5000 ppm are considered treatment-related, whereas the differences in water consumption between the low dose group and the control are considered to be without toxicological relevance.
 
BODY WEIGHT
In the F1 males, statistically significant reductions in mean body weights were seen in the highest dose group (5000 ppm) throughout the total study period (about 87% of the control value at the end of this study interval). Body weight gains of the 5000 ppm males, however, were generally similar to the respective control values. In total, the weight gain of the high dose F1 males was only about 5% lower than the body weight gain of the control males. Body weights of the 5000 ppm females were also statistically significantly reduced during the premating period (about 89% of the control value at the end of the premating phase). During gestation and lactation of F2 litter, mean body weights of the high dose F1 dams were statistically significantly lower than the corresponding control values. During premating, gestation and lactation periods body weight gain of the high dose females reached or even exceeded body weight gain of the controls.
The statistically significantly lower body weights recorded for the 5000 ppm F1 males and females were considered to be related to the test substance administration. A lower body weight was also recorded for these animals at the pup stage; during the following premating period the F1 parental animals of the high dose group gained substantially as much weight as the controls, but the body weights of the 5000 ppm rats were still reduced. All differences between the controls and 500 or 2500 ppm groups concerning body weights/weight gains, however, were regarded as spontaneous in nature.
 
MALE REPRODUCTIVE FUNCTION
For all F1 males which were placed with females to generate F2 pups, mating was confirmed. The male mating index was 100% in all groups.
 
FEMALE REPRODUCTIVE FUNCTION
The female mating index reached 100% in all groups. The mean duration until sperm was detected (day 0 pc) varied between 2.1 and 3.2 days and was highest for the control group, because one dam of this group had a prolonged cohabitation time. In the scheduled mating interval (F2), 2 control females did not become pregnant. Therefore, the fertility index was lowest in the control group (92%), whereas it was 100% in all substance-treated groups. There were no biologically relevant differences between test groups and the controls concerning the mean duration of gestation and the number of liveborn and stillborn F2 pups. All pregnant females - except one low dose female which had palpable pups in the uterus but did not deliver - gave birth to litters with liveborn pups. Consequently, the gestation and the live birth indices were not influenced by the administration of the test substance. The mean number of delivered pups/dam was not influenced by the test substance administered.

GROSS AND HISTOPATHOLOGICAL FINDINGS
- Thickening of the limiting ridge (margo plicatus) of the forestomach in most male and female rats.
- Minimal hyperkeratosis at the limiting ridge of the forestomach in most male and all female rats.
- Edema in the submucosa of the glandular stomach of 2 male and 10 female rats, minimal in all cases.


Dose descriptor:
NOAEL
Effect level:
240 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: general toxicity
Dose descriptor:
NOAEL
Effect level:
460 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: fertility
Remarks on result:
other: Generation: P and F1 (migrated information)
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
**** F1 GENERATION PUPS/LITTERS ****

VIABILITY
No substance-induced effects on pup mortality/viability were recorded during the lactation period. Both the viability index, as an indicator of the viability of the pups during the first 4 days after birth, and the lactation index, as an indicator how the pups were nursed during the rest of their rearing, do not show differences of biological relevance.
 
SEX RATIO
The sex distribution and sex ratios of live F1 pups on the day of birth and on day 21 post parturition (pp) did not show any substantial difference between controls and treated groups; all differences observed are regarded as spontaneous.
 
BODY WEIGHT
Mean body weights of F1 male and female pups were clearly reduced in 5000 ppm test group from day 14 pp onwards and impaired in the intermediate dose (2500 ppm) on day 21 pp when compared to the controls. On day 21 pp the pup weights (both sexes combined) in the high dose group were about 35% and those of the 2500 ppm pups about 11% lower than the corresponding control values. Body weight gains of the 2500 ppm and 5000 ppm pups were also statistically significantly decreased from days 7 (5000 ppm) or 14 pp (2500 ppm) up to weaning (day 21 pp). The reductions in pup body weights/body weight gains in the 5000 and 2500 ppm groups were attributed to the test substance administration. All other differences concerning pup body weights/body weight gains are without any biological relevance and lie within the biological range of variation.
 
CLINICAL OBSERVATIONS
- None of the F1 pups of any one group showed abnormal clinical findings during the lactation period.
- There were no biologically relevant differences between the control and the substance-treated F1 pups in the several morphological development stages monitored up to weaning.
- No remarkable differences between the groups were observed in the different behavioral tests which the pups underwent up to weaning.
- Only spontaneous findings were seen at necropsy (e.g. incisors sloped, hernia diaphragmatica, dilated renal pelvis) in very few of the pups examined. All findings were present in the concurrent control at a comparable frequency and/or did not show a clear relation to dosing.



**** F2 GENERATION PUPS/LITTERS ****

PUP NUMBER AND STATUS
The mean number of delivered F2 pups/dam in the treatment groups was similar to the relevant control value; moreover, the percentages of liveborn and stillborn F2 pups were comparable between the groups; all differences between the groups are in the range of biological variation.
 
VIABILITY/MORTALITY
During the lactation period a statistically significant increase in pups cannibalized by dams was noted in the high and intermediate dose groups. The increased cannibalization rate was predominantly caused by just one intermediate dose dam and two high dose dams. One Female of the high group, however, neglected her pups during the lactation period, thus nearly all pups of this dam died before schedule and/or were cannibalized. Occasionally insufficient nursing behavior and cannibalism occured also in control females, and thus the higher rate of cannibalized pups at these dose levels was not considered treatment-related.
There were no differences in biological relevance between the control and the 500, 2500 and 5000 ppm F2 pups concerning viability and mortality; consequently the viability and lactation indices were not affected by the test substance administration (although some statistically significant differences existed). All relevant values are inside the historical control range and/or do not show a clear relation to dosing; moreover it had to be taken into consideration, that the high dose dams delivered on average distinctly more pups (14.0 pups/dam) than the controls (12.9 pups/dam).
 
SEX RATIO
No remarkable differences between the control and the substance-treated groups were found in respect to the sex ratio of the F2 pups. The observable differences are in the range of biological variation.
 
BODY WEIGHT
Mean body weights/body weight gains of the F2 male and female pups of the 5000 ppm and 2500 ppm groups were clearly influenced by the test substance administration. Mean pup body weights of the 5000 ppm pups were statistically significantly lower than the corresponding control values from days 14 (males and females) until weaning on day 21 pp, when the high dose pups (both sexes combined) weighed about 32% less than the controls. Mean pup body weights of the 2500 ppm pups were statistically significantly (about 12%) lower than the corresponding control values on day 21 pp (both sexes combined). Weight gains of the pups of the 2500 and 5000 ppm test groups were also statistically significantly reduced from the second week of the lactation period onward, the reduction more pronounced in the 5000 ppm than in the 2500 ppm pups. All differences between the control group and the 500 ppm group concerning pup body weight data of the F2 generation were considered spontaneous in nature.
 
CLINICAL OBSERVATIONS
- F2 generation pups did not show any clinical signs up to weaning which could be attributed to the treatment. Hydrocephaly, which occurs also occasionally in control pups was recorded in one 500 ppm pup.
- Development stages: There was a statistically significantly lower incidence of F2 pups/litter with auditory canal opening on time in the intermediate dose group and with eye opening on time in the 5000 ppm group. The relevant values were within the historical control ranges and a clear relation to dosing was not observed. These effects must be considered in conjunction with the retarded weight gain of these pups and were therefore assessed as being possibly substance-related. There were no differences of biological relevance in different stages of development between the low dose and the control pups.
- No substantial differences could be noted between the F2 pups of all test groups and the control pups in the different behavioral tests. The observable differences were without biological relevance.
- The examinations of F2 pups at necropsy did not reveal any differences considered to be of biological relevance between the controls and the substance-treated groups either in the type or in the number of pup necropsy observations. A few pups of the different groups showed some spontaneous findings like hernia diaphragmatica, incisors sloped, dilated renal pelvis, hydroureter, hydrocephaly, focal liver necrosis, cardiomegaly, septal defect and post mortem autolysis.

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
53 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: general toxicity
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
53 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: general toxicity
Reproductive effects observed:
no

Mean Body Weight Changes (F0 parental animals), grams

Treatment week

 0 mg/kg bw/d

 53 mg/kg bw/d

 240 mg/kg bw/d

 460 mg/kg bw/d

male

female

male

female

male

female

male

female

0 - 1

52.6

23.8

52.2

24.8

52.3

23.6

46.7**

23.1

1 - 2

53.7

22.1

55.5

22.0

54.4

22.3

51.6

21.8

2 - 3

47.2

16.2

45.8

17.2

46.9

17.5

44.8

14.6

3 – 4

35.4

12.9

34.8

17.1*

35.1

14.9

32.4

17.8*

4 – 5

29.1

15.7

27.6

13.3

28.6

12.6

27.0

13.7

5 – 6

21.0

9.4

21.8

8.8

23.3

12.5

23.2

11.0

6 – 7

23.2

8.5

24.7

11.7

21.2

10.1

19.3*

10.7

7 – 8

20.4

7.7

17.7

10.3

19.8

7.8

18.5

8.6

8 – 9

19.3

9.8

19.1

7.1

18.0

7.2

16.6

8.1

9 – 10

13.1

4.7

13.0

4.5

14.6

7.9*

11.9

6.0

10 – 11

-7.0

 

-2.8

 

-2.8

 

-2.8

 

11 – 12

21.8

 

18.5

 

18.4

 

14.7**

 

12 – 13

14.8

 

13.3

 

13.7

 

11.1

 

13 – 14

8.0

 

10.7

 

9.7

 

6.1

 

14 – 15

9.2

 

7.0

 

7.5

 

8.1

 

15 – 16

10.5

 

10.4

 

8.0

 

9.9

 

16 – 17

5.7

 

7.9

 

7.3

 

4.6

 

17 – 18

2.7

 

4.6

 

1.7

 

1.4

 

18 – 19

1.1

 

0.2

 

1.3

 

1.6

 

19 - 20

4.1

 

6.7

 

5.8

 

5.0

 

*P<0.05

**P<0.01

Reproduction and litter data for F0 parents /F1 pups

 

0 ppm

500 ppm

2500 ppm

5000 ppm

Parents

Females mated

25

25

25

25

Females pregnant

24

23

23

24

Females with delivery

24

23

23

24

Mean duration of gestation (d)

22.0

21.9

21.9

21.9

Litter means

Live births/litter

13.8

13.8

13.7

14.3

Survivors day 4 preculling

13.3

13.5

13.4

13.8

Survivors day 4 postculling

7.5

7.9

7.9

7.9

Survivors day 21

7.5

7.9

7.8

7.8

Weight at day 1 (g) M/F

6.4/6.1

6.6/6.2

6.5/6.2

6.5/6.2

Weight at weaning (g) M/F

52.3/50.01

52.1/49.4

46.6**/44.6**

34.2**/32.7**

Sex ratio of live newborns % M/F

51/49

49/51

55/45

53/47

Selected as parents for the next generation M/F

25/25

25/25

25/25

25/25

**P=0.01

Reproduction and litter data for F1 parents /F2 pups

 

0 ppm

500 ppm

2500 ppm

5000 ppm

Parents

Females mated

25

25

25

25

Females pregnant

23

23

23

24

Females with delivery

23

23

23

24

Mean duration of gestation (d)

22.0

21.9

21.9

21.9

Litter means

Live births/litter

12.5

11.6

12.0

13.8

Survivors day 4 preculling

11.8

10.6

10.8

12.5

Survivors day 4 postculling

7.7

7.0

7.5

7.8

Survivors day 21

7.7

6.9

7.5

7.4

Weight at day 1 (g) M/F

6.3/5.9

6.5/6.2

6.1/5.8

6.4/6.1

Weight at weaning (g) M/F

50.4/48.4

52.1/49.4

44.6**/42.4**

34.5**/33.2**

Sex ratio of live newborns % M/F

47/53

55/45

53/47

49/51

**P=0.01

Development landmarks in the F2 (mean % of pups reaching criteria/litter)

Parameter

 

0 ppm

500 ppm

2500 ppm

5000 ppm

Historical control range

Pinna unfolding

93.6 (16.65)

93.5 (20.87)

80.7 (33.37)

86.9 (26.87)

74-100

Auditory canal opening

98.8 (3.87)

95.1 (20.90)

91.4*(18.09)

94.2 (12.26)

81-100

Eye opening

93.2 (18.08)

92.4 (21.89)

92.4 (21.89)

86.5*(21.15)

85-100

*P=0.05

Figures in parentheses indicate standard deviations.

Executive summary:

In a two-generation study according to OECD TG 416 acrylic acid was administered orally in the drinking water to male and female Wistar rats at doses of 0, 500, 2500, 5000 ppm (corresponding to approx. 53, 240, 460 mg/kg bw/day). At least 70 days after the beginning of treatment, F0 animals were mated to produce one litter (F1). Mating pairs were from the same dose group and F1 animals selected for breeding were continued in the same dosing group as their parents. Groups of 25 males and 25 females selected from F1 pups as F1 parental generation were offered drinking water containing 0, 500, 2500 and 5000 ppm of the test substance post weaning, and the breeding program was repeated to produce F2 litter. The study was terminated with the terminal sacrifice of the F2 weanlings and F1 adult animals.

The following results were observed (BAMM, 1994; Hellwig et al., 1997):

F0 generation:

460 mg/kg bw/day group:

- statistically significantly reduced food consumption in the males only during the first week of the premating period and in the females only during the second week of the lactation period,

- reduced water intake (11% - 13%) in both sexes during the premating period and in the females (12% - 14%) during gestation and lactation of F1 pups,

- impairment of body weights/body weight gains in males and - much less pronounced - in females,

- no treatment-related changes in organ weights,

- thickening of the limiting ridge (margo plicatus) of the forestomach in most male and female rats,

- minimal hyperkeratosis at the limiting ridge of the forestomach in most male and female rats,

- edema in the submucosa of the glandular stomach of most male (19/25) and female (14/25) rats, minimal in most cases.

In the two lower doses no treatment-related changes in clinical signs, organ weights and gross- and histopathological findings were recorded.

F1 generation:

460 mg/kg bw/day group:

- reduced body weights/body weight gains of the male and female pups,

- reduced food consumption (in the males and females) during the premating period and in the F1 dams during gestation and lactation,

- decrease in water consumption (18% - 27%) in both sexes during the premating period, and in the females (18% - 22%) during gestation and lactation,

- statistically significantly lower mean body weights compared to controls (both sexes ),

- no treatment-related changes in organ weights,

- thickening of the limiting ridge (margo plicatus) of the forestomach in most male and female rats,

- minimal hyperkeratosis at the limiting ridge of the forestomach in most male and all female rats,

- edema in the submucosa of the glandular stomach of 2 male and 10 female rats, minimal in all cases.

240 mg/kg bw/day group:

- marginally lower mean pup body weights compared to controls on day 21 p.p. and impaired body weight gains between days 14 - 21 p.p.,

- reduced water intake (9% - 13%) in both sexes during the premating phase (the differences in comparison to the control being statistically significant on most days), and in the females (6% - 13%) during gestation and lactation,

- no treatment-related changes in organ weights and gross- and histopathological findings.

At the lowest dose level no treatment-related changes in clinical signs, organ weights and gross- and histopathological findings were recorded.

F2 generation:

460 mg/kg bw/day group:

- reduced body weights/body weight gains (both sexes),

- lower incidence of pups/litter with eye opening on time.

240 mg/kg bw/day group:

- statistically significantly decreased body weight (about 12%) compared to the controls at the end of the lactation period; decreased pup body weight gains,

- fewer pups/litter with auditory canal opening on time.

At the lowest dose level no treatment-related changes in clinical signs, organ weights and gross- and histopathological findings were recorded.

It can be concluded from these results that the continuous administration of aqueous acrylic acid solutions to rats over two generations caused clear signs of toxicity in the highest dose group (5000 ppm =approx. 460 mg/kg body weight/day)in F0 and F1 parents. General toxicity was substantiated by e.g. reduced food and/or water consumption, impairment of body weights/body weight gains and gross and histopathological findings in the fore- and the glandular stomach (i.e. thickening of and minimal hyperkeratosis at the limiting ridge (margo plicatus), edema in the submucosa of the glandular stomach), which are a consequence of the administration of the acid solutions(indicative of the irritating properties of the test substance).

At 2500 ppm (= approx.240 mg/kg body weight/day) the water consumption of the F1 parental animals was still clearly reduced, but no further substance-related adverse effects on the parental rats were seen.

Clear adverse substance-induced effects were also noted for the progeny of the high dose of the F0 and F1. Impaired body weight/body weight gain in the F1 and F2 pups and some indications for delays in the morphological development of the F2 pups were seen. The latter finding was likely associated with the decreased body weight/body weight gain. Similar, but much less pronounced effects were also observed for the F1 and/or F2 pups at 2500 ppm.

500 ppm (= approx. 53 mg/kg body weight/day) were tolerated by both parental generations and their offspring without any changes which could be causally related to test substance administration.

Acrylic acid had no adverse effects on reproductive parameters of the parental animals of either generation (F0 and F1) of all groups (500, 2500 and 5000 ppm). No adverse effects on fertility and pre-implantation development could be detected; no effects on reproductive organs have been observed. The mating index of males in both generations and in all dose groups was 100 %. The fertility rate in the F0 generation was between 92-96 %; in the F1 generation in all dose groups the fertility rate was 100 %. The rate of pregnancy in both generations was not reduced. In both generations there were no differences in numbers of pups born alive.

Therefore, the NOAEL (no observed adverse effect level) with respect to reproductive function was 5000 ppm (=approx. 460 mg/kg body weight/day). The NOAEL with respect to general toxicity of the test substance was 2500 ppm (= approx.240 mg/kg body weight/day) for the F0 generation parental animals and 500 ppm (= approx. 53 mg/kg body weight/day)for the F1 males and females and the offspring (F1 and F2 pups).

 

Conclusion

 

In oral reproductive toxicity studies (rats) no effects on reproductive function (i.e. fertility) were observed.

Endpoint:
reproductive toxicity, other
Type of information:
other: Corap assessment
Adequacy of study:
weight of evidence
Reliability:
other: Substance evaluation under CoRAP
Principles of method if other than guideline:
2-Ethylhexan-1-ol was originally selected for substance evaluation in order to clarify suspected risks about:
- CMR properties, in particular developmental toxicity
Dose descriptor:
other: Corap evaluation
Remarks on result:
other: 2-EH is not a reproductive toxicant
Dose descriptor:
other: Corap evaluation
Remarks on result:
other: 2-EH is not a reproductive toxicant
Reproductive effects observed:
no
Conclusions:
The effects on the reproduction of 2-EH were evaluated in CoRAP in 2015:
•       The available data does not raise concern that 2-EH would affect fertility and sexual function.
Executive summary:

Excerpt from the CoRAP evaluation report:

Toxicity to reproduction (effects on fertility and developmental toxicity)

 

One of the reasons for the selection of 2-EH was health hazard concern. It was noted in the screening that developmental effects were observed in the pre-natal developmental toxicity studies. Therefore, a full evaluation of the available information was required in order to assess whether the observed developmental effects are the result of maternal toxicity. This part of the document reviews the available study reports in order to assess reproductive toxicity of 2-EH, and assess whether a proposal of harmonised C&L is needed for this endpoint.

Fertility

 

There is no study aimed at assessment of effect of 2-EH on fertility and sexual function on animal. Also observations on humans are not available.

 

However, taking into account that di (2 -ethylhexyl) terephthalate (DEHT) is metabolised to 2 -ethylhexan-1-ol (2-EH) and terephthalic acid, the 2- generation reproduction toxicity study of Faber et al. (2007) with DEHT provide some information on effect of 2- EH on fertility and sexual function. The 2 -ethylhexan-1 -ol and terephthalic acid are thus available in the body after di (2 -ethylhexyl) terephthalate (DEHT) application. Terephthalic acid has been shown to not affect fertility and sexual function (study report, 2003). Lack of effects of DEHT on fertility and sexual function found in the study of Faber et al. (2007) confirms findings of study report study (2003) and gives no indication that 2-EH would affect fertility and sexual function.

 

This conclusion is further supported by the results of repeated dose toxicity studies. The results of 90-day oral gavage study (Astil et al. , 1996) indicate that 2-EH at dose 250 and 500 mg/kg/day, thus comparable to the doses of 2-EH estimated in Faber et al. study (2007), does not affect morphology of testes or ovaries in rats and in mice. The relative weight of testes at a dose of 500 mg/kg/day was increased and that of ovaries decreased in rats only at dose 250 mg/kg /day, but not at a dose of 500 mg/kg. The relative weight of the testes (related to body weight) of 500 mg/kg/day group was slightly increased (5.5 % compared to control group). However, neither the absolute testes weight nor the relative weight of the testes related to brain weight did show any significant changes. Thus, the weight changes in testes can be attributed to the decreased body weight in males of high dose group (93 % of control value) and were not considered as adverse. There was decreased body weight gain in male and female rats at 500 mg/kg amounting to weight losses of 7% in males and 6% in females by Week 13. Due to low intensity, lack of dose response relationship and due to lack of histopathological changes in testes and ovaries, the changes in the weight of testes and ovaries alone are not regarded as adverse and do not warrant classification for fertility and sexual function. The weight of these reproductive organs were not affected in mice administered 2-EH by gavage at doses 250 and 500mg mg/kg, day for 13 weeks (Astil, 1996).

 

The effects of 2-EH on the testis are of interest because of the testicular atrophy and Sertoli cell damage produced in rats by high doses of DEHP, of which 2-EH is a metabolite (Gray and Gangolli, 1986). Sjoberg et al. (1986) showed that 350 mg of 2EH/kg/day administered to Sprague-Dawley (SD) rats by oral gavage for 5 days had no effect on testis weight and produced no effects on the seminiferous tubule. Similarly, concentrations of 0.2 mM of 2-EH were without effect on Sertoli cells from SD rats in primary culture studies (Gray and Beamond 1984). In the 13-week study in rats (Astil et al., 1996) there was a slight increase in relative testis weight at 500 mg/kg, not correlated with any morphological changes, and there were no gross or microscopic changes in testes of mice at 500 mg/kg. Therefore it is thus unlikely that the effects of DEHP on the testis are attributable to 2-EH.

The available data does not raise concern that 2-EH would affect fertility and sexual function.

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
28 Jul 2011
GLP compliance:
yes
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals: 70 days
- Basis for dose level selection: Dosage levels were selected based on the range-finding study. In that study, dosage levels of 40, 160, and 400 mg/kg/day were tested. Animals in the high-dosage group showed clinical signs (salivation and red material around the eyes, nose, and mouth), which were still observed to a lesser extent in the mid-dosage group, and the males were more affected than the females. Macroscopic examinations showed thickened and eroded stomach in the 400 mg/kg/day group males and thickened stomach was still observed in the 160 mg/kg/day group animals (predominantly in males). In the current study, the animals were dosed for a longer time period. Therefore, based on the results of the range-finding study, dosage levels of 20, 50, and 150 mg/kg/day were selected for the current study. The high-dosage level of 150 mg/kg/day was expected to show parental toxicity, whereas the low-dosage level of 20 mg/kg/day was not expected to show toxic effects.
- Inclusion/exclusion of extension of Cohort 1B: not performed as P0 and F1 Cohort 1A did not reveal any results
- Termination time for F2: not performed as P0 and F1 Cohort 1A did not reveal any results
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: not performed as P0 and F1 Cohort 1A did not reveal any results
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: not performed as P0 and F1 Cohort 1A did not reveal any results
- Route of administration: oral
Specific details on test material used for the study:
- Lot No. F534801GB
- Exp. date: 11 Dec 2016
- Colorless, clear liquid
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 8 weeks
- Weight at study initiation: Male body weights ranged from 264 g to 378 g and female body weights ranged from 154 g to 228 g on the initial day of test substance administration
- Housing: F0 parental animals were housed 2–3 per cage by sex in clean, solid-bottom cages with heat-treated aspen bedding material (Aspen Bed™ 1, Northeastern Products Corporation, Warrensburg, NY). Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly
- Diet: the basal diet was provided ad libitum throughout the acclimation period and during the study. The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5K96 Advanced Protocol® Verified Casein Diet 10 IF, was a certified feed with appropriate analyses performed by the manufacturer.
- Water: Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system was provided ad libitum throughout the acclimation period and during the study.
- Acclimation period: 24 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
other: 1% carboxymethylcellulose (medium viscosity), 0.014% Kolliphor® EL, and 0.0035% hydrochloric acid in deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance formulations were prepared daily as single formulations for each dosage level and maintained on wet ice, protected from light. The test substance formulations were stirred continuously on wet ice throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Concentration in vehicle: 0, 4, 10 and 30 mg/mL (corresponding to dosage levels of 0, 20 50 and 150 mg/kg/day)
- Amount of vehicle: 5 mL/kg
Details on mating procedure:
- M/F ratio per cage: The animals were paired on a 1:1 basis within each treatment group after a minimum of 70 days of treatment.
- Length of cohabitation: 14 day mating period
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Following positive evidence of mating, the F0 males and females were individually housed in solid-bottom cages with bedding material until the scheduled necropsy. The dams were housed in these cages with their litters until weaning on Lactation Day 21. Following weaning of the F1 litters, the weaned F1 pups were housed together by litter for 1 week. Beginning on PND 28, the F1 offspring were housed 2–3 per cage by sex in clean, solid-bottom cages with bedding material until necropsy. Females for which there was no evidence of mating were placed in clean, solid-bottom cages with bedding material upon completion of a 14-day mating period with no further opportunity for mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the 4, 10, and 30 mg/mL dosing formulations and from the middle stratum of the control group dosing formulations prepared on Study Day 0, 7, 15, and 30 for the first month and once a month thereafter (total of 9 sets). Analysis was performed using a validated gas chromatography method using flame ionization detection. The analysed dosing formulations were, with minor exceptions, within the range for suspensions (85% to 115%) and were homogeneous. Analysed concentrations above the acceptance criteria had no impact on the study as the no-observed-adverse-effect level (NOAEL) was determined to be 150 mg/kg/day, the highest dose evaluated on this study.
Duration of treatment / exposure:
The vehicle and test substance formulations were administered to the F0 males and females for a minimum of 70 consecutive days prior to mating. Dose administration for the F0 males continued throughout mating and through the day prior to euthanasia, for a total of 129-131 doses. The F0 females continued to be dosed throughout mating, gestation, and lactation, through the day prior to euthanasia, for a total of 132-136 doses. Animals selected for the F1 generation were directly administered the vehicle or test substance following weaning (beginning on PND 21) and continuing through the day prior to euthanasia (PND 91 [Cohort 1A] and between PND 103-110 [Cohort 1B]).
Frequency of treatment:
daily
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
30
Control animals:
yes, concurrent vehicle
Details on study design:
Dosage levels were selected based on the range-finding study (see any other information on materials and methods)
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS
All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS
Detailed physical examinations were recorded weekly, beginning 1 week prior to the initiation of test substance administration, for all parental animals throughout the study period. In addition, detailed physical examinations were conducted on Gestation Days 0, 7, 14, and 20 for all females with evidence of mating and on Lactation Days 1, 7, 14, and 21. In addition, animals were observed for signs of toxicity approximately 1 hour following dose administration. Special attention was paid to the degree of salivation and lacrimation, presence or absence of urination and defecation (including polyuria and diarrhoea), pupil size, degree of palpebral closure, presence of convulsions, tremors, or abnormal movements, presence of posture and gait abnormalities, the presence of any unusual or abnormal behaviours, and any repetitive actions (stereotypies). Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labour, delayed labour) or other difficulties. In addition, the social groups were observed at the appropriate intervals for findings that could not be attributed to a single animal.

BODY WEIGHT
Individual F0 male body weights were recorded weekly, beginning 1 week prior to the initiation of test substance administration, throughout the study and prior to the scheduled necropsy. Individual F0 female body weights were recorded weekly, beginning 1 week prior to the initiation of test substance administration, until evidence of copulation was observed. Mean weekly body weights and body weight changes are presented for each interval. In addition, cumulative mean body weight changes are presented for the pre-mating treatment period (males and females) and for the entire F0 treatment period (males only). Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 1, 4, 7, 10, 14, 17, and 21. After weaning (Lactation Day 21), weekly body weights were recorded for these females until the scheduled necropsy. For F0 females with no evidence of mating, weekly body weights were recorded until necropsy.

FOOD CONSUMPTION
F0 male and female food consumption was measured weekly, beginning 1 week prior to the initiation of test substance administration and continuing until cohabitation. Food consumption was measured on a per cage basis for the corresponding body weight intervals. Food consumption was normalized to the number of animals/cage and was reported as g/animal/day. Food intake was not recorded during the breeding period. Following the breeding period, individual food consumption for males and for females with no evidence of mating was measured on a weekly basis until the scheduled necropsy. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 1, 4, 7, 10, 14, 17, and 21. Food intake was reported for the corresponding intervals of gestation and lactation, and also for Gestation Days 0–20 and Lactation Days 1–21. Food efficiency (body weight gained as a percentage of food consumed) was also calculated

CLINICAL PATHOLOGY
Blood and urine samples for clinical pathology evaluations (haematology, coagulation, serum chemistry, and urinalysis) were collected prior to the scheduled necropsy (Study Days 129-136 for the F0 males and females). All animals were fasted overnight prior to blood collection while in metabolism cages for urine collection blood and urine samples were collected from 10 randomly selected F0 animals/sex/group sent to necropsy. Blood for serum chemistry and haematology was collected from the jugular vein using the hand-held technique following anaesthesia with isoflurane inhalation. Blood for coagulation parameters was collected from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation.

- Haematology and coagulation parameters examined: Total leukocyte count (WBC), Erythrocyte count (RBC), Haemoglobin (HGB), Haematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (Platelet), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Reticulocyte count Percent (RETIC), Absolute (RETIC Absolute), Mean platelet volume (MPV), Differential leukocyte count Percent and absolute, Neutrophil (NEU), Lymphocyte (LYMPH), Monocyte (MONO), Eosinophil (EOS), Basophil (BASO), Large unstained cell (LUC), Red cell distribution width (RDW), Haemoglobin distribution width (HDW), Platelet estimate, Red cell morphology (RBC Morphology).
- Serum chemistry parameters examined: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio) [by calculation], Total bilirubin (Total BILI), Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyl transferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides (Triglyceride), Appearance, Bile Acids
- Urinalysis parameters examined: Specific gravity (SG), pH, Urobilinogen (URO), Total volume (TVOL), Colour (COL), Clarity (CLA), Protein (PRO), Glucose (GLU)

THYROID HORMONE ANALYSIS
Blood (approximately 1.5 mL) for thyroid hormone levels was collected via the retro-orbital sinus following isoflurane anaesthesia from F0 males and females (10/sex/group) following the completion of weaning of all F0 females
- Parameters evaluated: Thyroxine (T4) and Thyroid Stimulating Hormone (TSH)
Oestrous cyclicity (parental animals):
Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the oestrous cycle of each F0 female for 14 days prior to cohabitation and continuing until evidence of mating was observed or until the end of the mating period. The average cycle length was calculated for complete oestrous cycles (i.e., the total number of returns to metoestrous [M] or dioestrous [D] from oestrus [E] or prooestrous [P], beginning 14 days prior to initiation of the mating period and continuing until the detection of evidence of mating). Oestrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the mean individual oestrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the oestrous cycle. At the end of the study, the overall pattern of each female was characterized as regularly cycling, irregularly cycling, not cycling, or insufficient data.
Sperm parameters (parental animals):
Immediately upon euthanasia, the reproductive tract of each F0 male was exposed via a ventral mid-line incision. The right epididymis was excised and weighed separately. An incision was made in the distal region of the right cauda epididymis. The right cauda epididymis was then placed in Dulbecco's phosphate-buffered saline (maintained at approximately 37°C) with 10 mg/mL BSA. After a 10 to 20-minute incubation period, a sample of sperm was loaded onto a slide with a 100-µm chamber depth for determination of sperm motility. Because sperm motility can be affected by temperature shock, all slides and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37°C). Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyser. The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported:
Percent Motile (or Progrressively Molite) Sperm = Number of Motile (or Progressively Motile) Sperm / Total Number of Sperm Counted x 100

The right epididymis was then placed in modified Davidson’s solution for subsequent microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique. Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded. The left testis and cauda epididymis from all F0 males from the control and high-dose groups were weighed, stored frozen, homogenized, and analysed for determination of homogenization resistant spermatid count and calculation of sperm production rate. An aliquot of each sample was added to a solution containing a DNA-specific fluorescent dye (the dye stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20-µm chamber depth. Illumination from a xenon lamp within the analyser allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample. The sperm production rate was calculated as follows:
Sperm Production Rate = No. of Sperm Per Gram of Tissue / 6.1 days
6.1 days = The rate of turnover of the germinal epithelium
Litter observations:
STANDARDISATION OF LITTERS
To reduce variability among the litters, 10 pups/litter, 5 pups/sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 10 pups. All selections were performed by computerized randomization. Following selection, culled pups were euthanized by decapitation (those pups used for blood/thyroid collection) or an intraperitoneal injection of sodium pentobarbital, and examined macroscopically; tissues with gross lesions were preserved in 10% neutral-buffered formalin.

PARAMETERS EXAMINED PRE-WEANING
- A daily record of litter size was maintained. Intact offspring that were found dead during PND 0–4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels.7 Pups found dead on PND 0 or 1 had the lungs removed and placed in a saline-filled jar. If the lungs sank to the bottom of the jar, the pup had no prior documentation of being viable, and there was no evidence of milk in the stomach, the pup was considered to be stillborn. If the lungs floated, the pup was considered to be found dead on the originally documented day (PND 0 or 1).
- Litters were examined daily for survival and any adverse changes in appearance or behaviour. Each pup received a detailed physical examination on PND 1, 4, 7, 14, and 21.
- Pups were individually weighed on PND 1, 4 (before culling), 7, 14, and 21.
- Pups were individually sexed on PND 0, 4 (before culling), 7, 14, and 21.
- The anogenital distance of all F1 pups was measured on PND 1
- On PND 12, all F1 male offspring were evaluated for the presence of thoracic nipples/areolae.

GROSS EXAMINATION OF DEAD PUPS
A detailed gross necropsy was performed on any pup found dead after PND 4 and prior to weaning. Tissues were preserved for possible future histopathological examination only as deemed necessary by the gross findings.

POST-WEANING DEVELOPMENTAL LANDMARKS
- Balanopreputial Separation: Each male was observed for balanopreputial separation beginning on PND 35. The age at which balanopreputial separation was first observed was recorded for each pup. Examination of the pups continued daily until balanopreputial separation was present. Body weights were recorded at the age of attainment of this landmark. In addition, the appearance of a partial and complete balanopreputial separation or a persistent thread of tissue between the glans and prepuce was recorded.
- Vaginal Patency: Each female was observed for vaginal perforation beginning on PND 25. The age at which the vaginal lumen was first observed to open was recorded for each pup. Examination of the females was continued daily until vaginal patency was present. Body weights were recorded at the age of attainment of this landmark. In addition, the appearance of a small “pin hole”, a vaginal thread, and complete vaginal opening were recorded.
- Oestrous Cyclicity (Cohort 1A): Beginning on the day vaginal opening was observed, vaginal lavages were performed daily for all F1 females assigned to Cohort 1A and the slides were evaluated microscopically to determine the stage of the estrous cycle of each female until the first sign of oestrus (cornified cells) was observed. The age of first vaginal oestrus after vaginal opening was recorded. Vaginal lavages were also performed daily for all F1 females assigned to Cohort 1A and the slides were evaluated microscopically to determine the stage of the oestrous cycle of each female for 2 weeks during PND 75–91 (the day of necropsy). The average cycle length was calculated and reported for complete oestrous cycles (i.e., the total number of returns to metaoestrus [M] or dioestrus [D] from oestrus [E] or prooestrus [P]). Oestrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. At the end of the study, the overall pattern of each female was characterized as regularly cycling, irregularly cycling, not cycling, or insufficient data.

POST WEANING OBSERVATIONS
- Clinical observations: Following weaning, all animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity. Detailed physical examinations were performed weekly until necropsy. In addition, animals were observed for signs of toxicity approximately 1 hour following dose administration.
- Body weights: F1 males and females were weighed weekly following weaning, and on the day of euthanasia.
- Food Consumption: F1 male and female food consumption was measured weekly, beginning on PND 28, until the day prior to euthanasia. Food consumption was normalized to the number of animals/cage and was reported as g/animal/day. Food efficiency (body weight gained as a percentage of food consumed) was also calculated and reported for these intervals.

CLINICAL PATHOLOGY (Cohort 1A)
Blood and urine samples for clinical pathology evaluations (haematology, coagulation, serum chemistry, and urinalysis) were collected prior to the scheduled necropsy (PND 91 for the Cohort 1A F1 males and females). All animals were fasted overnight prior to blood collection while in metabolism cages for urine collection blood and urine samples were collected from10 randomly selected F0 and F1 Cohort 1A animals/sex/group sent to necropsy. Blood for serum chemistry and haematology was collected from the jugular vein using the hand-held technique following anaesthesia with isoflurane inhalation. Blood for coagulation parameters was collected from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation.

- Haematology and coagulation parameters examined: Total leukocyte count (WBC), Erythrocyte count (RBC), Haemoglobin (HGB), Haematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (Platelet), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Reticulocyte count Percent (RETIC), Absolute (RETIC Absolute), Mean platelet volume (MPV), Differential leukocyte count Percent and absolute, Neutrophil (NEU), Lymphocyte (LYMPH), Monocyte (MONO), Eosinophil (EOS), Basophil (BASO), Large unstained cell (LUC), Red cell distribution width (RDW), Haemoglobin distribution width (HDW), Platelet estimate, Red cell morphology (RBC Morphology).
- Serum chemistry parameters examined: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio) [by calculation], Total bilirubin (Total BILI), Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyl transferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides (Triglyceride), Appearance, Bile Acids
- Urinalysis: Specific gravity (SG), pH, Urobilinogen (URO), Total volume (TVOL), Colour (COL), Clarity (CLA), Protein (PRO), Glucose (GLU)
Postmortem examinations (parental animals):
SACRIFICE
The surviving F0 males and females were euthanized on Study Days 129-136

GROSS NECROPSY
- A complete necropsy was conducted on all F0 animals that were found dead, euthanized in extremis, or at termination. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system. Clinical findings that were verified at necropsy were designated CEO. For F0 females that delivered, the numbers of former implantation sites (the attachment site of the placenta to the uterus) were recorded. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. Numbers of corpora lutea were also recorded for females necropsied during gestation through Lactation Day 4. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.
- The following F0 parental tissues and organs were collected and were placed in 10% neutral-buffered formalin: Adrenal glands (2), Aorta (1), Bone with marrow (sternebrae), Brain (7 levels), Coagulating glands (2), Eyes with optic nerve (2) [a], Gastrointestinal tract, Oesophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys (2), Lacrimal/Harderian glands (2), Liver (sections of 2 lobes fixed and also 2 sections frozen) [b], Lungs (including bronchi, fixed by inflation with fixative) (5), Lymph node (mandibular and mesenteric) (1), Ovaries [c] and oviducts (2), Pancreas (1), Peripheral nerve (tibial), Pituitary (1), Prostate (1), Mandibular salivary gland (1), Seminal vesicles (2), Skeletal muscle (rectus femoris) (1), Skin with mammary gland and subcutis (1)[d], Spinal cord (cervical, thoracic, lumbar) (3), Spleen (1), Testes with epididymides (1) and, vas deferens (1) [e,f],Thymus, Thyroids with trachea [g] [with parathyroids if present (1)], Urinary bladder, uterus with cervix and vagina (4), All gross lesions (all groups).
The number in parentheses is the minimum number of sections to be examined if the tissue was chosen for histopathologic examination.
[a] Eyes with optic nerves were fixed in Davidson’s solution.
[b] Sections of 2 liver lobes were placed in 10% neutral-buffered formalin and 2 sections of liver lobes were flash frozen in liquid nitrogen and stored frozen at -70°C for possible future analysis.
[c] Ovaries were fixed in 10% neutral-buffered formalin for approximately 48 hours following which all ovaries were transferred to 70% ethanol. The ovaries were processed to blocks.
[d] For females, a corresponding section of skin was taken from the same anatomic area for males.
[e] Testis (right only) and epididymides (right and left caput and body) were fixed in modified Davidson’s solution. Care was taken to ensure separation between the left and right organs.
[f] If the left testis or epididymis was noted with a gross lesion, the left testis and epididymis were fixed in modified Davidson’s solution and the right testis and epididymis were homogenized
[g] Thyroids and parathyroids (if present) were removed from the trachea following fixation to ensure tissue structure was maintained during prosection.

ORGAN WEIGHTS
Except as noted, paired organs were weighed together. Absolute weights and organ to final body weight and brain weight ratios were reported. The following organs were weighed from all F0 animals at the scheduled necropsies: Adrenal glands, Brain, Epididymides (total and cauda) [a], Heart, Kidneys, Levator ani and bulbocavernosus (LABC) muscle group, Liver, Ovaries, Pituitary gland, Prostate gland, Seminal vesicles with coagulating glands (with accessory fluids), Spleen, Testes [a], Thyroids with parathyroids [b], Thymus gland, Uterus with oviducts and cervix.
[a] These paired organs were weighed separately.
[b] Tissues were weighed after fixation in 10% neutral-buffered formalin.

HISTOPATHOLOGY
Microscopic examination was performed on all tissues from all F0 parental from the control and high-dose groups and for all adult animals found dead and euthanized in extremis. In addition, reproductive organs of all F0 animals suspected of reduced fertility (e.g., those that failed to mate, conceive, sire, or deliver healthy offspring, or for which oestrous cyclicity or sperm number, motility, or morphology were affected) were subjected to histopathological examination. Additionally, all gross lesions were examined microscopically, irrespective of group.
Postmortem examinations (offspring):
SACRIFICE
- Euthanasia was scheduled PND 91 [Cohort 1A] and between PND 103-110 [Cohort 1B]
- All nonselected F1 weanlings were euthanized on PND 21.
- These animals were subjected to post-mortem examinations (macroscopic and microscopic examination) as follows:

GROSS NECROPSY
- A complete necropsy was conducted on all F1 animals in Cohorts 1A and 1B that were found dead, euthanized in extremis, or at termination. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system. Clinical findings that were verified at necropsy were designated CEO.
- The following F1 Cohorts 1A and 1B tissues and organs were collected and were placed in 10% neutral-buffered formalin: Adrenal glands (2), Aorta (1), Bone with marrow (sternebrae), Brain (7 levels), Coagulating glands (2), Eyes with optic nerve (2) [a], Gastrointestinal tract, Oesophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys (2), Lacrimal/Harderian glands (2), Liver (sections of 2 lobes fixed and also 2 sections frozen) [b], Lungs (including bronchi, fixed by inflation with fixative) (5), Lymph node (mandibular and mesenteric) (1), Ovaries [c] and oviducts (2), Pancreas (1), Peripheral nerve (tibial), Pituitary (1), Prostate (1), Mandibular salivary gland (1), Seminal vesicles (2), Skeletal muscle (rectus femoris) (1), Skin with mammary gland and subcutis (1)[d], Spinal cord (cervical, thoracic, lumbar) (3), Spleen (1), Testes with epididymides (1) and, vas deferens (1) [e,f],Thymus, Thyroids with trachea [g] [with parathyroids if present (1)], Urinary bladder, uterus with cervix and vagina (4), All gross lesions (all groups).
The number in parentheses is the minimum number of sections to be examined if the tissue was chosen for histopathologic examination.
[a] Eyes with optic nerves were fixed in Davidson’s solution.
[b] Sections of 2 liver lobes were placed in 10% neutral-buffered formalin and 2 sections of liver lobes were flash frozen in liquid nitrogen and stored frozen at -70°C for possible future analysis.
[c] Ovaries were fixed in 10% neutral-buffered formalin for approximately 48 hours following which all ovaries were transferred to 70% ethanol. The ovaries were processed to blocks.
[d] For females, a corresponding section of skin was taken from the same anatomic area for males.
[e] Testis (right only) and epididymides (right and left caput and body) were fixed in modified Davidson’s solution. Both testes and epididymides from animals euthanized in extremis and from all males in Cohort 1B were fixed in modified Davidson’s solution. Care was taken to ensure separation between the left and right organs.
[f] If the left testis or epididymis was noted with a gross lesion, the left testis and epididymis were fixed in modified Davidson’s solution and the right testis and epididymis were homogenized
[g] Thyroids and parathyroids (if present) were removed from the trachea following fixation to ensure tissue structure was maintained during prosection.
- Gross necropsies with emphasis on developmental morphology and organs of the reproductive system were performed on all nonselected F1 pups euthanized on PND 21.
-The following tissues/organs from 10 F1 nonselected pups/sex/group were retained in 10% neutral-buffered formalin for possible histopathologic examination: Brain, Mammary gland, Spleen, Thymus, Thyroids, All gross lesions

ORGAN WEIGHTS
- Except as noted, paired organs were weighed together. Absolute weights and organ to final body weight and brain weight ratios were reported. The following organs were weighed from all F1 animals in Cohort 1A at the scheduled necropsies: Adrenal glands, Brain, Epididymides (total and cauda) [a,c], Heart, Kidneys, Levator ani and bulbocavernosus (LABC) muscle group[c], Liver, Ovaries [c], Pituitary gland [c], Prostate gland [c], Seminal vesicles with coagulating glands (with accessory fluids) [c], Spleen, Testes [a,c], Thyroids with parathyroids [b], Thymus gland, Uterus with oviducts and cervix [c]
[a] These paired organs were weighed separately.
[b] Tissues were weighed after fixation in 10% neutral-buffered formalin.
[c] Also weighed for F1 Cohort 1B animals.
- The brain, spleen and thymus were weighed from 10 nonselected F1 pups/sex/group on PND 21. In addition, the thyroids were weighed (after fixation) from the same 10 pups/sex/group that were used for blood collection

HISTOPATHOLOGY
- Microscopic examination was performed on all tissues from all F1 Cohort 1A animals from the control and high-dose groups and for all adult animals found dead and euthanized in extremis. Additionally, all gross lesions were examined microscopically, irrespective of group.

SPERM PARAMETERS (Cohort 1A)
Immediately upon euthanasia, the reproductive tract of each F1 Cohort 1A male was exposed via a ventral mid-line incision. The right epididymis was excised and weighed separately. An incision was made in the distal region of the right cauda epididymis. The right cauda epididymis was then placed in Dulbecco's phosphate-buffered saline (maintained at approximately 37°C) with 10 mg/mL BSA. After a 10 to 20-minute incubation period, a sample of sperm was loaded onto a slide with a 100-µm chamber depth for determination of sperm motility. Because sperm motility can be affected by temperature shock, all slides and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37°C). Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyser. The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported:
Percent Motile (or Progrressively Molite) Sperm = Number of Motile (or Progressively Motile) Sperm / Total Number of Sperm Counted x 100

The right epididymis was then placed in modified Davidson’s solution for subsequent microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique. Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded. The left testis and cauda epididymis from all F1 Cohort 1A males from the control and high-dose groups were weighed, stored frozen, homogenized, and analysed for determination of homogenization resistant spermatid count and calculation of sperm production rate. An aliquot of each sample was added to a solution containing a DNA-specific fluorescent dye (the dye stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20-µm chamber depth. Illumination from a xenon lamp within the analyser allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample. The sperm production rate was calculated as follows:
Sperm Production Rate = No. of Sperm Per Gram of Tissue / 6.1 days
6.1 days = The rate of turnover of the germinal epithelium
Statistics:
see any other information on materials and methods
Reproductive indices:
- Male Mating Index (%) = No. of Males with Evidence of Mating / Total No. of Males Used for Mating x 100
- Female Mating Index (%) = No. of Females with Evidence of Mating or Females Confirmed Pregnant / Total No. of Females Used for Mating x 100
- Male Fertility Index (%) = No. of Males Siring a Litter/ Total No. of Males Used for Mating x 100
- Male Copulation Index (%) = No. of Males Siring a Litter/ No. of Males with Evidence of Mating (or Females Confirmed Pregnant) x 100
- Female Fertility Index (%) = No. of Females with Confirmed Pregnancy/ Total No. of Females used for Mating x 100
- Female Conception Index (%) = No. of Females with Confirmed Pregnancy/ No. of Females with Evidence of Mating (or Females Confirmed Pregnant) x 100
Offspring viability indices:
- Mean Live Litter Size = Total Viable Pups on PND 0/ No. Litters with Viable Pups on PND 0
- Postnatal Survival Between Birth and PND 0 or PND 4 (Pre-Selection)(% Per Litter) = Sum of (Viable Pups/Litter on PND 0 or PND 4 [Pre-Selection]/No. of Pups Born/Litter)/ No. of Litters/Group x 100
- Postnatal Survival for All Other Intervals (% Per Litter) = Sum of (Viable Pups/Litter at End of Interval N/Viable Pups/Litter at Start of Interval N)/ No. of Litters/Group x 100

Where N = PND 0–1, 1–4 (Pre-Selection), 4 (Post-Selection)–7, 7–14, 14–21, Birth to PND 4 (Pre-Selection), or 4 (Post-Selection)–21
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related clinical observations were noted during the F0 generation at the daily examinations, weekly detailed physical examinations, and/or approximately 1 hour following dose administration. Clonic convulsions were noted for 1 male in the 20 mg/kg/day group, 1 female in the 50 mg/kg/day group, and 1 male and 1 female in the 150 mg/kg/day group sporadically throughout the treatment period; this finding was noted at the daily examinations, weekly detailed physical examinations, at the time of dose administration, and/or approximately 1 hour following dose administration. This finding was transient and was not observed in the F1 generation, and therefore was not considered test substance-related. Other observations noted in the test substance-treated groups, including red and/or yellow material, scabbing, and/or hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test substance-related effects on survival were noted for F0 males and females at any dosage level. One female (in the 50 mg/kg/day group) was euthanized in extremis on Gestation Day 26 due to poor general health. The cause of morbidity was the acute inflammation of the uterus observed microscopically and the partially decomposed fetuses observed grossly. Because all animals in the high-dose group (150 mg/kg/day) survived to the scheduled necropsy, the moribundity in the 50 mg/kg/day group was not considered test substance-related. All other F0 parental animals in the control, 20, 50, and 150 mg/kg/day groups survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- No test substance-related effects on F0 mean body weights, body weight gains, and cumulative body weight gains were noted in the 20, 50, and 150 mg/kg/day groups. Differences from the control group were not statistically significant, with the following exceptions. For F0 males, significantly (p < 0.01) lower mean body weight gains were noted in the 20 mg/kg/day group during Study Days 112–119 and in the 150 mg/kg/day group during Study Days 91–98 compared to the control group; these differences were transient, did not occur in a dose-related manner, and/or were not of sufficient magnitude to affect the entire generation interval (Study Days 0–126) or mean body weights in these groups, and therefore were not considered test substance-related. A significantly (p < 0.05) higher mean body weight was noted for the 150 mg/kg/day group F0 females compared to the control group on Study Day 14; this difference was transient and no corresponding effect on mean body weight gain was noted in this group, and therefore was not considered test substance-related.
- Mean F0 maternal body weights, body weight gains, and cumulative body weight gains were unaffected by test substance administration during gestation. Differences between the control, 20, 50, and 150 mg/kg/day groups were slight and not statistically significant, with the following exception. A significantly (p < 0.05) lower mean body weight gain was noted in the 50 mg/kg/day group F0 females compared to the control group during Gestation Days 17–20; this difference was transient, did not occur in a dose-related manner, was not of sufficient magnitude to affect the overall gestation treatment interval (Gestation Days 0–20) or mean body weights in this group, and therefore was not considered test substance-related.
- Mean F0 maternal body weights, body weight gains, and cumulative body weight gains were unaffected by test substance administration during lactation. Differences between the control, 20, 50, and 150 mg/kg/day groups were slight and not statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
- Mean F0 food consumption in the 20, 50, and 150 mg/kg/day groups was unaffected by test substance administration. The values in the test substance-treated groups were generally similar to the control group values for the pre-mating period (females) or the entire generation (males). Differences from the control group were not statistically significant, with the following exceptions. In F0 females, significantly (p < 0.05 or p < 0.01) higher mean food consumption was noted in the 20 mg/kg/day group during Study Days 7–14 and in the 150 mg/kg/day group during Study Days 0–7 and 14–21; these differences were transient, did not occur in a dose-related manner, and/or were not of sufficient magnitude to affect mean body weights at these dosage levels, and therefore were not considered test substance-related.
- Mean F0 maternal food consumption was unaffected by test substance administration during gestation. Differences between the control, 20, 50, and 150 mg/kg/day groups were slight and not statistically significant.
- Mean F0 maternal food consumption was unaffected by test substance administration during lactation. Differences between the control, 20, 50, and 150 mg/kg/day groups were slight and not statistically significant.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
- Mean F0 food efficiency in the 20, 50, and 150 mg/kg/day groups were unaffected by test substance administration.
- Differences from the control group were not statistically significant, with the following exceptions. Significantly (p < 0.01) lower mean food efficiency was noted for F0 males in the 20 mg/kg/day group during Study Days 112–119 and in the 150 mg/kg/day group during Study Days 91-98 compared to the control group; these differences were transient, did not occur in a dose-related manner, and/or a corresponding effect on mean food consumption was not noted at these dosage levels, and therefore they were not considered test substance-related.
- Mean F0 maternal food efficiency was unaffected by test substance administration during gestation.
- Mean F0 maternal food efficiency was unaffected by test substanceadministration during lactation.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related alterations in serum chemistry parameters. Significantly (p < 0.05) higher mean serum albumin was observed in the 150 mg/kg/day group F0 females when compared to the concurrent control group. The mean and individual animal values were within the Charles River Ashland historical control ranges for female rats. The finding was considered a result of individual animal variation rather than a true test substance-related change.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related microscopic findings were noted in the nonglandular stomach and liver of the 150 mg/kg/day group F0 males and females and the kidneys in the 150 mg/kg/day group F0 females.
- Nonglandular stomach findings were limited to minimal to moderate epithelial hyperplasia and hyperkeratosis. The hyperplasia was characterized by increased thickness (increased number of cell layers) of the squamous epithelium with an increased thickness of the keratinized oute layers of the epithelium (hyperkeratosis). In one 150 mg/kg/day group F0 female, minimal edema and congestion were observed in the submucosa adjacent to the hyperplasia and hyperkeratosis. These changes were not associated with any clinical pathology changes. Although considered an adaptive change, the mild to moderate severity of the hyperplasia and/or hyperkeratosis in the F0 generation were considered adverse in the 150 mg/kg/day group males and females.
- Liver changes observed microscopically included increased incidence of minimal to mild biliary hyperplasia in the 150 mg/kg/day group F0 males and females and minimal to mild randomly scattered hepatocellular necrosis in the 150 mg/kg/day group F0 males. Biliary hyperplasia was characterized by increased numbers of small bile duct profiles in the portal triads. Biliary hyperplasia can occur as a normal age related finding in rats and was observed in the concurrent control group in this study. The severity was minimal to mild and a clinical pathology correlate for the finding was not observed. Similarly, randomly distributed hepatocellular necrosis can be observed spontaneously in rats and was observed in the control group in this study. The severity of the necrosis was minimal to mild and the change lacked clinical pathology correlates. There was no association of the biliary hyperplasia to the necrosis. Changes observed in the liver were considered nonadverse.
- Increased severity of mineralization at the corticomedullary junction of the kidneys was observed in the 150 mg/kg/day group F0 females. This increase in severity (minimal to mild) did not result in alterations to the clinical pathology parameters related to renal function and were considered nonadverse.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on serum levels of T4 (thyroxine) or TSH (thyroid stimulating hormone) in F0 males or females. Differences from the control group were slight, did not occur in a dose-related manner, and/or were not statistically significant.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The mean lengths of oestrous cycles in the test groups were also similar to the control group value. None of these differences were statistically significant. All females were noted to be cycling, and the percentage of females cycling regularly was similar across all groups.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any dosage level. Differences from the control group were slight and were not statistically significant, with the following exceptions. Left cauda epididymal sperm concentration was significantly (p < 0.05) lower in the 150 mg/kg/day group compared to the control group. However, there were no effects on reproductive performance (mating and fertility) and no other effects on spermatogenic endpoints at this dosage level; therefore, the lower epididymal sperm concentration noted at 150 mg/kg/day was not considered test substance-related. Significantly (p < 0.05) lower mean motility and progressive motility were noted in the 20 mg/kg/day group compared to the control group; these differences did not occur in a dose-related manner, and therefore were not considered test substance-related.
Reproductive performance:
no effects observed
Description (incidence and severity):
No test substance-related effects on F0 reproductive performance were observed at any dosage level. The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value.
No test substance-related effects were noted on mean gestation lengths or the process of parturition at any dosage level.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no reproductive effects observed
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: local effects (nonglandular stomach)
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by test substance administration.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No test substance-related effects on survival were noted for F1 animals at any dosage level. In the 50 mg/kg/day group, 1 female was found dead on PND 82 following clinical observations of gasping prior to and following dose administration on the day of death. Based on the findings noted for this female at necropsy (esophageal perforation and clear fluid contents in the thoracic cavity [approximately 8.0 mL]), the cause of death was determined to be intubation error, and therefore was not considered test substance-related. In the control group, 1 male was euthanized in extremis on PND 28 due to clinical observations of head tilt and circling on the day of euthanasia; at necropsy, this male was noted with dilated lateral ventricles in the brain. This correlated to the marked chronic active inflammation in the brain. All other F1 animals in the control, 20, 50, and 150 mg/kg/day groups survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- Mean F1 male and female pup body weights and body weight changes in the 20, 50, and 150 mg/kg/day groups were unaffected by test substance administration throughout the postnatal period.
- No test substance-related effects on mean body weights, body weight gains, and cumulative body weight gains were noted in the 20, 50, and 150 mg/kg/day group F1 males and females. Differences from the control group were slight and not statistically significant, with the following exception. A significantly (p < 0.01) lower mean body weight gain was noted in the 20 mg/kg/day group F1 males compared to the control group during PND 42-49; this difference was transient, did not occur in a dose-related manner, and was not of sufficient magnitude to affect mean body weights at this dosage level, and therefore was not considered test substance-related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Mean F1 food consumption in the 20, 50, and 150 mg/kg/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant, with the following exceptions. Significantly (p < 0.05 or p < 0.01) lower mean food consumption was noted in the 20 mg/kg/day group F1 males during PND 42–49 and 63–70, in the 50 mg/kg/day group F1 males during PND 70–77, and in the 50 mg/kg/day group F1 females during PND 49–63 and 70–77 compared to the control group; these differences were transient, did not occur in a dose-related manner, and were not of sufficient magnitude to affect mean body weights at these dosage levels, and therefore were not considered test substance-related.
Food efficiency:
no effects observed
Description (incidence and severity):
Mean F1 food efficiency in the 20, 50, and 150 mg/kg/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
- There were no test substance-related effects on serum chemistry parameters.
Urinalysis findings:
no effects observed
Sexual maturation:
no effects observed
Description (incidence and severity):
- Mean estrous cycle lengths in the test substance-treated groups were similar to the control group.
- No test substance-related effects were observed on F1 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any dosage level.
- There were no test substance-related effects on ovarian primordial follicle counts in the 150 mg/kg/day group F1 females in Cohort 1A compared to the control group.
- Mean ages of attainment of balanopreputial separation and mean body weights at the age of attainment were unaffected by test substance administration.
- Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by test substance administration.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- No test substance-related effects on organ weights (absolute, relative to final body weight, and relative to brain weight) were observed for PND 21 pups at any dosage level.
-There were no test substance-related changes in organ weights for F1 males and females in Cohort 1A. The absolute and relative pituitary weights were significantly (p < 0.05 or p < 0.01) higher in the 50 and 150 mg/kg/day group F1 females when compared to the concurrent control. These values fell within the historical control reference ranges, were not associated with any microscopic findings, and/or were considered a result of individual animal variation rather than a true test substance-related effect.

-No test substance-related effects on organ weights (absolute and relative to final body weight) were observed at any dosage level for F1 animals in Cohort 1B (necropsied on PND 103–110). Differences from the control group were slight and not statistically significant, with the following exceptions. A higher mean absolute left testis weight was noted in 50 mg/kg/day group F1 males and higher mean relative (to final body weight) left and right testis weights were noted in the 20 and 50 mg/kg/day group F1 males compared to the control group; these differences did not occur in a dose-related manner, and therefore were not considered test substance-related.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- The numbers of pups (litters) found dead from PND 0 through the selection of the F1 generation were 50(13), 46(14), 24(5), and 31(11) in the control, 20, 50, and 150 mg/kg/day groups, respectively. No internal findings that could be attributed to parental administration of the test substance were noted at the necropsies of pups that were found dead. Aside from the presence or absence of milk in the stomach and lungs that did or did not float, internal findings included renal papilla(e) not fully developed (Woo and Hoar Grade 1) for 2 pups in the 50 mg/kg/day group and red fluid contents in the bladder for 1 pup in the 20 mg/kg/day group. Because the findings in the 20 and 50 mg/kg/day groups were noted infrequently and in a non-dose-related manner, they were not considered test substance-related.
- No internal findings that could be attributed to parental administration of the test substance were noted at the necropsy of culled pups euthanized on PND 4. Internal findings included the developmental variations of distended ureters noted for one pup in the 50 mg/kg/day group and hemorrhagic ring around the iris noted for 1 pup each in the control and 20 mg/kg/day groups. These findings occurred in single pups and in a non-dose-related manner, and therefore were not considered test substance-related.
- No internal findings that could be attributed to parental administration of the test substance were noted at the necropsy of pups euthanized on PND 21. Dilated renal pelvis was noted for 1, 2 , and 1 pups in the 20, 50, and 150 mg/kg/day groups, respectively; 1 of these same pups in the 50 mg/kg/day group was also noted with distended ureters. In the 150 mg/kg/day group, 1 pup was noted with opacity of the eyes. Foamy contents in the trachea was noted for groups, respectively; this finding was also noted for 1 pup in the control group. A short tail was noted for 1 pup each in the control and 50 mg/kg/day groups. In the 50 mg/kg/day group, 1 up was noted with yellow areas on the liver and 1 Pup was noted with a fractured tail. Dark red discoloration of the eyes was noted for 1 pup each in the control and 20 mg/kg/day groups; 11 pup was also noted with a small left eye. The aforementioned findings were noted insingle pups or litters, occurred infrequently or similarly in the control group, and/or did not occur in a dose-related manner, and therefore were not considered test substance-related.
- At the PND 21 necropsy of F1 weanlings selected for hormone analysis, no internal findings that could be attributed to parental administration of the test substance were noted at any dosage level. Internal findings were limited to dark red discoloration on the thyroid glands for a Male Pup in the 20 mg/kg/day group and an accessory spleen for a Female Pup in the 150 mg/kg/day group. Because these findings were limited to a single fetus and/or did not occur in a dose-related manner, they were not considered test substance-related.
- Thickened stomachs were observed in the 20, 50, and 150 mg/kg/day group F1 males and the 150 mg/kg/day group F1 females in Cohort 1A and were considered test substance-related. At the scheduled F1 male and female necropsies for Cohort 1B, thickened stomachs were noted in the 50 and 150 mg/kg/day group F1 males and in the 150 mg/kg/day group F1 females and were considered test substance-related; given the lack of systemic toxicity
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related microscopic changes were observed in the nonglandular stomach for F1 males and females in Cohort 1A at all dosage levels. Hyperkeratosis was observed in all test substance-administered groups while epithelial hyperplasia was observed in the 50 and 150 mg/kg/day groups. The findings were not associated with clinical pathology changes but were of similar, although slightly less, severity when compared to the F0 generation and were considered adverse in the 150 mg/kg/day group males and females.
There were no test substance-related effects on ovarian primordial follicle counts in the 150 mg/kg/day group F1 females in Cohort 1A compared to the control group.
Other effects:
no effects observed
Description (incidence and severity):
- No test substance-related effects on anogenital distance were noted for F1 pups in the 20, 50, and 150 mg/kg/day groups. Longer anogenital distances (absolute and relative to the cube root of pup body weight) were noted for F1 male pups in the test substance-treated groups compared to the control group; differences were generally significant (p < 0.05 or p < 0.01). However, the aforementioned differe ces did not occur in a dose-related manner and the mean absolute values in the 20, 50, and 150 mg/kg/day groups (4.07 mm, 4.19 mm, and 4.17 mm, respectively) were within the range of values in the Charles River Ashland historical.
- Areolae/nipple anlagen in the F1 male pups was not affected by test substance administration. There was no retention of nipples noted in any male pup on study on PND 12.
- There were no test substance-related changes in total T4 or TSH in pups that were culled on PND 4.
- There were no test substance-related changes in serum T4 or TSH in F1 weanlings on PND 21.
- There were no test substance-related changes in serum T4 or TSH in F1 animals assigned to Cohort 1A.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: local effects (nonglandular stomach)
Critical effects observed:
no
Reproductive effects observed:
no
Executive summary:

The objective of this study was to evaluate the potential effects of the test substance, n-butyl acrylate, on reproduction in an extended 1-generation study covering life stages that are not covered by other types of toxicity studies. This included determining the effects of n-butyl acrylate on reproductive endpoints that require the interaction of males with females, females with conceptus and females with offspring, and the F1 generation until after sexual maturity. This study provided information about the effects of n-butyl acrylate on the integrity and performance of the adult male and female reproductive systems and included an evaluation of the F1 offspring for potential effects of n-butyl acrylate on the developing nervous systems and alterations in endocrine function (including thyroid perturbations).

Three groups of male and female Crl:CD(SD) rats (30/sex/group) were administered the test substance, n-butyl acrylate, daily by oral gavage for at least 70 consecutive days prior to mating. Dosage levels were 20, 50, and 150 mg/kg/day for the F0 and F1 generations. A concurrent control group of 30 rats/sex/group received the vehicle (1% carboxymethylcellulose [medium viscosity], 0.014% Kolliphor® EL, and 0.0035% hydrochloric acid in deionized water) on a comparable regimen. F0 animals were approximately 8 weeks of age at the initiation of test substance administration. The F0 males and females continued to receive the test substance throughout mating, during gestation and lactation for females, and through the day prior to euthanasia (Study Days 129-136). Offspring (2 pups/sex/litter from all available litters) from the pairing of the F0 animals were selected on PND 21 to constitute the F1 generation. The test substance was administered to the offspring selected to become the F1 generation following weaning (beginning on PND 21). F1 animals were further subdivided into cohorts such that a minimum of 20 pups/sex/group were assigned to Cohort 1A or 1B for reproductive/developmental toxicity testing. F1 animals continued to receive the test substance through the day prior to euthanasia. All animals were observed twice daily for appearance and behavior. Clinical observations, detailed physical examinations, body weights, and food consumption were recorded at appropriate intervals for F0 males and females throughout the study (including during gestation and lactation for F0 females). Vaginal lavages were performed daily for F0 females for determination of estrous cycles beginning 14 days prior to cohabitation. All F0 females were allowed to deliver and rear their pups until weaning on Lactation Day 21. Blood samples for thyroid hormone evaluation were collected and clinical pathology evaluations (urinalysis, hematology, coagulation, and serum chemistry) were performed for all F0 animals at the scheduled euthanasia. Complete necropsies were conducted on all F0 males and females (Study Days 129-136); selected organs were weighed. Spermatogenic endpoints were recorded for all F0 males, and ovarian primordial follicle counts were recorded for all F0 females suspected of reduced fertility. Designated tissues were examined microscopically. Clinical observations, body weights, and sexes for F1 pups were recorded at appropriate intervals and anogenital distance was recorded on PND 1. On PND 4, litters were culled to 5 pups/sex/litter; culled pups were examined macroscopically and blood samples for thyroid hormone analysis were collected. All F1 male pups were evaluated for areolae/nipple anlagen on PND 12. Prior to PND 21, 2 pups/sex/litter from all available litters (=20 litters/group) were selected to remain on study for further reproductive/developmental toxicity testing. Nonselected F1 pups were necropsied on PND 21; blood samples for thyroid hormone analysis were collected and selected organs were weighed. Clinical observations, body weights, and food consumption were recorded at appropriate intervals for the selected F1 males and females following weaning. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated. The selected F1 animals were randomly assigned to 1 of 2 cohorts (Cohorts 1A or 1B): Cohort 1A females were evaluated for estrous cyclicity. Blood samples for thyroid hormone analysis were collected from F1 males and females assigned to Cohort 1A on PND 85. Clinical pathology evaluations (urinalysis, hematology, coagulation, and serum chemistry) were performed for F1 animals in Cohort 1A at the scheduled euthanasia. F1 animals in Cohorts 1A and 1B were necropsied on PND 91 or between PND 103-110, respectively; selected organs were weighed. Spermatogenic endpoints were recorded for all F1 males in Cohort 1A, and ovarian primordial follicle counts were recorded for F1 females in Cohort 1A. Designated tissues from F1 animals in Cohort 1A were examined microscopically.

There were no test substance-related effects on survival for F0 and F1 animals at any dosage level. One F0 female in the 50 mg/kg/day group was euthanized in extremis on Gestation Day 26 due to poor general health; in the absence of moribundity at the highest dosage level, the moribundity in the 50 mg/kg/day group was not considered test substance-related. In the F1 generation, 1 female in the 50 mg/kg/day group was found dead on PND 82; based on the macroscopic findings for this female noted at necropsy (esophageal perforation and clear fluid contents in the thoracic cavity), the cause of death was determined to be intubation error and therefore was not considered test substance-related. One F1 male in the control group was euthanized in extremis on PND 28 following adverse clinical findings (head tilt and circling). All other F0 and F1 males and females survived to the scheduled necropsies. No test substance-related clinical observations were noted for F0 and F1 animals at any dosage level. Mean body weights, body weight gains, food consumption, and food efficiency in the 20, 50, and 150 mg/kg/day F0 and F1 males and females were unaffected by test substance administration. No test substance-related effects were noted on F0 reproductive performance (male and female mating and fertility, male copulation, and female conception indices), the mean number of days between pairing and coitus, mean gestation lengths, or the process of parturition. In addition, there were no test substance-related effects on F0 or F1 estrous cyclicity or spermatogenic parameters (testicular and epididymal sperm concentrations, sperm production rate, sperm motility, and sperm morphology) at any dosage level. There were no test substance-related effects on the number of F1 pups born, live litter size, percentage of males at birth, F1 postnatal survival, clinical observations, anogenital distance, offspring body weights, necropsy findings, or developmental landmarks (areolae/nipple retention, vaginal patency, and balanopreputial separation). No test substance-related effects on clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were noted for F0 and F1 animals at any dosage level. In addition, no test substance-related effects on serum levels of T4 (thyroxine) or TSH (thyroid stimulating hormone) were noted in F0 and F1 males or females or F1 pups (on PND 4 and 21).

Test substance-related histologic changes were observed in all dosage groups in the F0 generation and F1 males and females in Cohort 1A. Epithelial hyperplasia and/or hyperkeratosis was observed in the nonglandular stomach in all test substance-treated groups examined. Mild to moderate changes in the 150 mg/kg/day group males and females of the F0 and F1 generations were considered adverse in this study. Microscopic changes in the stomach were associated with the gross observation of thickened nonglandular stomach, but were not associated with any clinical pathology, organ, or body weight changes. In the F0 generation, a nonadverse increased incidence of biliary hyperplasia (males and females) and random hepatocellular necrosis (males) were observed in the liver in the 150 mg/kg/day group. Additionally, nonadverse test substance-related microscopic findings (increased severity of mineralization at the corticomedullary junction) were observed in the kidneys of the 150 mg/kg/day group F0 females. Thickened stomachs were noted in the 50 and 150 mg/kg/day group F1 males and in the 150 mg/kg/day group F1 females at the scheduled necropsies for Cohort 1B; this finding was considered test substance-related and adverse in the 150 mg/kg/day group males and females. No other test substance-related internal findings were observed at any dosage level for F1 Cohort 1B animals. No test substance-related effects on the mean number of F0 implantation sites or number of unaccounted-for sites were noted at any dosage level. No test substance-related macroscopic findings were observed in F1 pups that were found dead, culled on PND 4, or examined at the scheduled necropsy on PND 21; F1 pup organ weights on PND 21 were unaffected by test substance administration. No test substance-related effects on ovarian primordial follicle counts were noted in the F0 females suspected of reduced fertility or F1 Cohort 1A females. There were no test substance-related effects on organ weights noted for F0 and F1 males and females at any dosage level.

Due to the absence of systemic toxicity noted for F0 and F1 males and females throughout the study, a dosage level of 150 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 and F1 male and female systemic toxicity when n-butyl acrylate was administered orally by gavage to Crl:CD(SD) rats. Epithelial hyperplasia and/or hyperkeratosis in the nonglandular stomach noted in the 150 mg/kg/day group F0 and F1 males and females were considered adverse; based on these results, 50 mg/kg/day was considered to be the NOAEL and 150 mg/kg/day was considered to be the lowest-observed-adverse-effect level (LOAEL) for local effects in the F0 and F1 generations. Based on the lack of effects noted for F1 litters, a dosage level of 150 mg/kg/day was considered to be the NOAEL for neonatal toxicity. There was no evidence of reproductive toxicity at any dosage level based on evaluation of reproductive performance in the F0 generation and sperm measurements and estrous cyclicity in the F0 and F1 generations. Therefore, the NOAEL for F0 and F1 reproductive toxicity was considered to be 150 mg/kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
998 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
Reliability 1; EOGRTS according to OECD TG 443 under GLP
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

2-Ethylhexyl acrylate

An OECD TG #443 as requested in the Compliance Check Decision number: CCH-D-2114340871-51-01/F, was performed - draft report available.

2-Ethylhexyl acrylate - Extended One-Generation Reproduction Toxicity Study in Wistar Rats Administration via the Diet without extension to produce the F2 generation according to OECD TG 443 (ARTF 2023)

(detailed description see key information above)

2-Ethylhexyl acrylate was administered to groups of 25 male and 25 female healthy young Wistar rats as a homogeneous addition to the food in different concentrations (0, 1500, 5000 and 12500 ppm). F0 animals were treated at least for 10 weeks prior to mating to produce a litter (F1 generation). Mating pairs were from the same dose group. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 2 different cohorts (1A and 1B) which were subjected to specific postweaning examinations.

The study was terminated with the terminal sacrifice of the F1 rearing animals of cohort 1A. Test diets containing 2-Ethylhexyl acrylate were offered continuously throughout the study.

Under the conditions of the present extended one-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 5000 ppm (about 357 mg/kg bw/d in males, 453 mg/kg bw/d in females) in the F0 parental and the F1 adolescent/adult rats, based on evidence for local toxicity in the gastrointestinal tract which conveyed to systemic toxicity such as decreased body weight/body weight gain across gener-

ations and cohorts, at the LOAEL (Lowest Observed Adverse Effect Level) of 12500 ppm (about 998 mg/kg bw/d in males, 1136 mg/kg bw/d in females).

The NOAEL for fertility and reproductive performance is 12500 ppm (about 998 mg/kg bw/d in males, 1136 mg/kg bw/d in females), the highest dose tested

The NOAEL for developmental toxicity in the F1 progeny is 12500 ppm (about 998 mg/kg bw/d in males, 1136 mg/kg bw/d in females), the highest dose tested.

The NOAEL for developmental immunotoxicity for the F1 progeny is 12500 ppm (about 998 mg/kg bw/d in males, 1136 mg/kg bw/d in females), the highest dose tested.

 

2 -Ethylhexanol

The toxicity to reproduction (effects on fertility and developmental toxicity) of 2-ethylhexanol was evaluated by the eMS Poland as a part of the substance evaluation process under the REACH Regulation (EC) No 1907/2006.

One of the reasons for the selection of 2-EH was health hazard concern. It was noted in the screening that developmental effects were observed in the pre-natal developmental toxicity studies. Therefore, a full evaluation of the available information was required in order to assess whether the observed developmental effects are the result of maternal toxicity. This part of the document reviews the available study reports in order to assess reproductive toxicity of 2-EH, and assess whether a proposal of harmonised C&L is needed for this endpoint.

Excerpt from the CoRAP evaluation report (2015) related to the fertility:

There is no study aimed at assessment of effect of 2-EH on fertility and sexual function on animal. Also observations on humans are not available.

However, taking into account that di (2-ethylhexyl) terephthalate (DEHT) is metabolised to 2-EH and terephthalic acid, the 2- generation reproduction toxicity study of Faber et al. (2007) with DEHT provide some information on effect of 2-EH on fertility and sexual function. The 2-EH and terephthalic acid are thus available in the body after di (2-ethylhexyl) terephthalate (DEHT) application. Terephthalic acid has been shown to not affect fertility and sexual function (study report, 2003). Lack of effects of DEHT on fertility and sexual function found in the study of Faber et al. (2007) confirms findings of study report study (2003) and gives no indication that 2-EH would affect fertility and sexual function.

 

This conclusion is further supported by the results of repeated dose toxicity studies. The results of 90-day oral gavage study (Astil et al., 1996) indicate that 2-EH at dose 250 and 500 mg/kg/day, thus comparable to the doses of 2-EH estimated in Faber et al. study (2007), does not affect morphology of testes or ovaries in rats and in mice. The relative weight of testes at a dose of 500 mg/kg/day was increased and that of ovaries decreased in rats only at dose 250 mg/kg /day, but not at a dose of 500 mg/kg. The relative weight of the testes (related to body weight) of 500 mg/kg/day group was slightly increased (5.5 % compared to control group). However, neither the absolute testes weight nor the relative weight of the testes related to brain weight did show any significant changes. Thus, the weight changes in testes can be attributed to the decreased body weight in males of high dose group (93 % of control value) and were not considered as adverse. There was decreased body weight gain in male and female rats at 500 mg/kg amounting to weight losses of 7% in males and 6% in females by Week 13. Due to low intensity, lack of dose response relationship and due to lack of histopathological changes in testes and ovaries, the changes in the weight of testes and ovaries alone are not regarded as adverse and do not warrant classification for fertility and sexual function. The weight of these reproductive organs were not affected in mice administered 2-EH by gavage at doses 250 and 500mg mg/kg, day for 13 weeks (Astil et al., 1996).

The available data does not raise concern that 2-EH would affect fertility and sexual function.

 

Acrylic acid

Possible effects on reproductive performance were investigated by oral administration (via drinking water) in two different studies with rats.

In a one-generation study with F344 rats (DePass, 1983), the animals (10 males and 20 females per dose group) received acrylic acid at dose levels corresponding to 0, 83, 250 or 750 mg/kg bw/d for 13 weeks. Each male was then mated with 2 females and exposure continued for both sexes throughout gestation and lactation. Dose-related reductions in food and water consumption and consequently in body weight gain were observed in the F0 animals, most pronounced and statistically significant at the 750 mg/kg bw/d dose level. In the high-dose group, pups of both sexes showed decreased body weight gain. Also in F0 males of the high-dose group a reduction in absolute and relative liver weights and in F0 females a reduction in both absolute and relative spleen weights was observed. At the high-dose level the fertility index of males and females, the gestation index, the number of pups born alive and the percentage of pups weaned were numerically, but not statistically significantly reduced. However, the data should be interpreted cautiously because of a relatively atypical control group, in which the female fertility index and the mean number of pups born alive/litter were reduced compared to the historical control of the testing laboratory. Therefore, based on the above findings the maximum dosage level that did not produce a deleterious reproductive effect for one generation of exposure of acrylic acid in the drinking water of F344 rats was estimated to be 250 mg/kg bw/day (= NOAEL for reproductive effects in the F0 and F1 generation).

 

In a two-generation study (OECD 416) acrylic acid was administered orally (in drinking water) to Wistar rats at doses of 0, 500, 2,500, 5,000 mg/l (53, 240, 460 mg/kg bw/d) (Hellwig, 1994). The following results were observed: in the male F0 parental generation there were no signs of general toxicity. In the female parental generation reduction of food and drinking water consumption were observed at 5000 mg/l during the period of pregnancy. Dose dependent reduction of food and drinking water consumption during the lactation period were observed at 2,500 mg/l. In the F1 generation in both sexes dose-dependent reduction of food and drinking water consumption at 2,500 mg/l were observed. Body weight and body weight gain in both sexes were reduced. But in both sexes of the F0 and F1 generation there were no abnormal clinical signs. No adverse effects on fertility and pre-implantation development could be detected; no effects on reproductive organs have been observed. The mating index of males in both generations and in all dose groups was 100%. The fertility rate in the F0 generation was between 92-96%; in the F1 generation in all dose groups the fertility rate was 100%. The rate of pregnancy in both generations was not reduced. In both generations there were no differences in numbers of pups born alive. The NOAEL for reproductive function was 460 mg/kg bw/d. The NOAEL for general toxicity was 240 mg/kg bw/d for the F0 generation, but 53 mg/kg bw/d for the F1 generation.

 

n-Butyl acrylate

For n-butyl acrylate an extended one generation study according to OECD 443 and GLP was performed. 30 Crl:CD(SD) rats were exposed to 20, 50 and 150 mg/kg bw/day by oral (gavage) exposure route (Herberth, 2017).

There were no test substance-related effects on survival for F0 and F1 animals at any dosage level. No test substance-related clinical observations were noted for F0 and F1 animals at any dosage level. Mean body weights, body weight gains, food consumption, and food efficiency in the 20, 50, and 150 mg/kg/day F0 and F1 males and females were unaffected by test substance administration. No test substance-related effects were noted on F0 reproductive performance (male and female mating and fertility, male copulation, and female conception indices), the mean number of days between pairing and coitus, mean gestation lengths, or the process of parturition. In addition, there were no test substance-related effects on F0 or F1 estrous cyclicity or spermatogenic parameters (testicular and epididymal sperm concentrations, sperm production rate, sperm motility, and sperm morphology) at any dosage level.

There were no test substance-related effects on the number of F1 pups born, live litter size, percentage of males at birth, F1 postnatal survival, clinical observations, anogenital distance, offspring body weights, necropsy findings, or developmental landmarks (areolae/nipple retention, vaginal patency, and balanopreputial separation).

No test substance-related effects on clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were noted for F0 and F1 animals at any dosage level. In addition, no test substance-related effects on serum levels of T4 (thyroxine) or TSH (thyroid stimulating hormone) were noted in F0 and F1 males or females or F1 pups (on PND 4 and 21). Test substance-related histologic changes were observed in all dosage groups in the F0 generation and F1 males and females in Cohort 1A. Epithelial hyperplasia and/or hyperkeratosis was observed in the nonglandular stomach in all test substance-treated groups examined. Mild to moderate changes in the 150 mg/kg/day group males and females of the F0 and F1 generations were considered adverse in this study. Microscopic changes in the stomach were associated with the gross observation of thickened nonglandular stomach, but were not associated with any clinical pathology, organ, or body weight changes. In the F0 generation, a nonadverse increased incidence of biliary hyperplasia (males and females) and random hepatocellular necrosis (males) were observed in the liver in the 150 mg/kg/day group. Additionally, nonadverse test substance-related microscopic findings (increased severity of mineralization at the corticomedullary junction) were observed in the kidneys of the 150 mg/kg/day group F0 females. Thickened stomachs were noted in the 50 and 150 mg/kg/day group F1 males and in the 150 mg/kg/day group F1 females at the scheduled necropsies for Cohort 1B; this finding was considered test substance-related and adverse in the 150 mg/kg/day group males and females. No other test substance-related internal findings were observed at any dosage level for F1 Cohort 1B animals. No test substance-related effects on the mean number of F0 implantation sites or number of unaccounted-for sites were noted at any dosage level. No test substance-related macroscopic findings were observed in F1 pups that were found dead, culled on PND 4, or examined at the scheduled necropsy on PND 21; F1 pup organ weights on PND 21 were unaffected by test substance administration. No test substance-related effects on ovarian primordial follicle counts were noted in the F0 females suspected of reduced fertility or F1 Cohort 1A females. There were no test substance-related effects on organ weights noted for F0 and F1 males and females at any dosage level.

Due to the absence of systemic toxicity noted for F0 and F1 males and females throughout the study, a dosage level of 150 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 and F1 male and female systemic toxicity when n-butyl acrylate was administered orally by gavage to Crl:CD(SD) rats. Epithelial hyperplasia and/or hyperkeratosis in the nonglandular stomach noted in the 150 mg/kg/day group F0 and F1 males and females were considered adverse; based on these results, 50 mg/kg/day was considered to be the NOAEL and 150 mg/kg/day was considered to be the lowest-observed-adverse-effect level (LOAEL) for local effects in the F0 and F1 generations. Based on the lack of effects noted for F1 litters, a dosage level of 150 mg/kg/day was considered to be the NOAEL for neonatal toxicity. There was no evidence of reproductive toxicity at any dosage level based on evaluation of reproductive performance in the F0 generation and sperm measurements and estrous cyclicity in the F0 and F1 generations. Therefore, the NOAEL for F0 and F1 reproductive toxicity was considered to be 150 mg/kg/day.

 

The design of the OECD TG#443 study was based on the results of a range finding study performed following a protocol adapted from the OECD TG# 421 (Herberth, 2016). n-Butyl Acrylate was administered daily by oral gavage to Sprague Dawley rats for a minimum of 28 consecutive days for males, females were dosed daily for 14 days prior to mating, throughout mating, and continuing until euthanasia (lactation day 4 for females that delivered, post-mating day 25, or post-cohabitation day 25 for females that did not deliver). Treatment was administered at dose levels of 0, 40, 160, and 400 mg/kg/day.

There were no test substance related early deaths. Clinical findings (males and females), lower mean body weights and food consumption (males) were noted at 400 mg/kg/day. There was test substance-related higher spleen weights (absolute and relative to brain and final body weights) in the 400 mg/kg/day group males. There was test substance-related thickened nonglandular stomach in the 160 and 400 mg/kg/day group males and 160 mg/kg/day group females, as well as test substance-related erosion of the nonglandular stomach in the 400 mg/kg/day group males. Microscopically, there was test substance-related hyperkeratosis and epithelial hyperplasia of the nonglandular stomach in all treated groups. Additionally, there was test substance-related mixed cell inflammation in all treated males and in 400 mg/kg/day group females, and ulceration in the nonglandular stomach in the 400 mg/kg/day group males. Finally, there was test substance-related mixed cell inflammation in the glandular stomach of the 400 mg/kg/day group males. . No effects were noted on male and female reproductive performance or the growth, viability, and clinical condition of the F1 generation.

 

Additional references:

Astill BD, Deckardt K, Gembardt C, Gingell R, Guest D, Hodgson JR, Mellert W, Murphy SR and Tyler TR, (1996). Prechronic toxicity studies on 2-ethylhexanol in F334 rats and B6C3F1 mice. Fundamental and Applied Toxicology, 29, 31-39.

Faber WD, Deyo JA, Stump DG, Ruble K (2007). Two-Generation Reproduction Study of Di-2-Ethylhexyl Terephthalate in Crl: Birth Defects Res B Dev Reprod Toxicol. 2007 Apr;80(2):69-81.

 

Effects on developmental toxicity

Description of key information

An OECD TG #414 in rabbits as requested in the Compliance Check Decision number: CCH-D-2114340871-51-01/F, was performed - draft report available.

2-Ethylhexyl acrylate - Prenatal Developmental Toxicity Study in New Zealand White Rabbits Administration in the diet (ARTF 2023)

In a prenatal developmental toxicity study, 2-Ethylhexyl acrylate was administered to pregnant New Zealand White rabbits daily as a homogeneous addition to the food from GD 6-29 at target dose levels of 50, 150 and 300 mg/kg bw/d.
Analyses confirmed the correctness of the prepared concentrations, their homogeneous distribution and the stability of the test substance in the vehicle.
The average test substance intake was 39, 119 and 191 mg/kg bw/d, based on all females proven to be pregnant during the treatment period. The reason for the discrepancy to the target doses was reduced food consumption which could not be compensated by applying the procedure described, which involved the adaptation of the test substance
concentration in the diet to the actual food intake.
However, cases of reduced food consumption were rather irregularly distributed among the individual rabbits within the treatment groups. It is notable, that all females experiencing abortions or complete litter losses had very long time periods of either food refusal or distinctly reduced food consumption, which in turn had a profound influence on average test substance intake of the respective treatment group.

These were the affected animals:

Animal Day of abortion Average food consumption (g/animal/day) Effective dose (test substance intake, mg/kg bw/d)
50 mg/kg bw/d      
27 GD 26 2.5 1.0
32 GD 20 2.2 0.9
48 GD 18 3.8 1.6
150 mg/kg bw/d      
56 GD 22 6.0 7.3
66   2.6 2.7
69   2.9 3.3
300 mg/kg bw/d      
76 GD 27 11.7 26.9
80 GD 23 5.7 7.2
81 GD 28 30.8 68.0
83 GD 28 3.7 9.3
96   2.4 5.4

Considering the very low effective dose levels in these animals, it is rather unlikely that these abortions/resorptions were caused by the test substance. They were clearly a consequence of the food refusal of these individuals which happened independent of the applied test substance concentrations in the diet, as the relatively equal distribution of the cases about the dose groups shows.
If these outliers were discounted from the calculation of test substance intake, the corresponding group means were about 42 mg/kg bw/d, 131 mg/kg bw/d and 225 mg/kg bw/d for the low-, mid- and high dose group, respectively. Corresponding to similar values for food consumption, the difference to the target doses in the low- and mid-dose groups amounted to -15% and -13%, which is acceptable considering the complex manufacturing and feeding processes (see sections 2.2.3. and 3.5.1.). In contrast, the test substance intake in high-dose group was still about 25% below target, which corresponded to about 30% lower food consumption, and indicates some food refusal which was consistently spread across all remaining animals in the group. This is likely to be a sign that the animals began to experience the irritating potential of the test item in the stomach which, considering the consequences for (net) body weight change and carcass weights, can be referred to as an adverse effect.

The irritating potential of the test item in the stomach is known from other studies, particularly in rats, e.g. in BASF study 91R0903/00S063 (OECD 443) where similar concentrations in the diet caused a high incidence of erosion/ulcer in the stomach, particularly, in female animals. Instinctive reduction of consumption of medicated diet prevented the rabbits from acquiring similar pathological findings. In the OECD 443, where animals were dosed up to the limit dose no signs of systemic toxicity were detected and no effects on hormone parameters and no outward signs of developmental toxicity in any of the animals were observed . The over-riding toxic effect of the low alkyl acrylates, including 2EHA, is the local toxicity at the PoE (see also repeated dose toxicity chapter 5.4), which can also be seen in relation with the rapid metabolism (see also chapter toxicokinetics). None of the lower acrylates exhibited reproduction or developmental toxicity, as such the likelihood of inducing adverse developmental effects at higher doses which would be sufficient for classification of the substance is extremely low.


Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 300 mg/kg bw/d.
Regarding pathology, all findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

There were no differences of biological relevance between the control and the substance treated groups (50, 150 and 300 mg/kg bw/d) in the reproductive parameters, such as conception rate, mean number of corpora lutea, total implantations, resorptions and live fetuses, fetal sex ratio or in the values calculated for the pre- and the post-implantation losses.
Litter losses in two females of the mid-dose group (Nos. 66 and 69) and one female of the high-dose group (No. 96) were associated with extensive periods of food refusal by these animals (see above) and were regarded as not being related to a potential developmental toxicity of the test compound.
Number and type of fetal external, soft tissue and skeletal findings, which were classified as malformations and/or variations, did not show any differences of toxicological relevance between the groups and/or were present the historical control range of the test facility.

The test substance is considered not teratogenic in rabbits under the conditions employed in this study.

CONCLUSION

Under the conditions of this prenatal developmental toxicity study, the oral administration of 2 -Ethylhexyl acrylate as homogeneous inclusion in the diet to pregnant New Zealand White rabbits from implantation to the expected day of parturition (GD 6-29) caused evidence indicative of maternal toxicity (food refusal because of stomach irritation) at the highest dose. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is effectively 119 mg/kg bw/d.

Since there was no evidence for treatment-related adverse effects of the test substance on fetal morphology the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is effectively 191 mg/kg bw/d.

It is notable, that a number of females across all dose groups experienced abortions or complete litter losses after very long time periods of either food refusal or distinctly reduced food consumption. These effects occurred independent of the applied test substance concentrations in the diet and had a profound influence on average test substance intake of their respective treatment groups. If these outliers were discounted from the calculation of test substance intake (= effective dose), the corresponding group means were about 42 mg/kg bw/d, 131 mg/kg bw/d and 225 mg/kg bw/d for the low-, mid- and high dose group, respectively.

The test substance is considered not teratogenic in rabbits under the conditions employed in this study.

2 -Ethylhexyl acrylate - OECD 414 in rats: Relative Developmental Toxicities of  Acrylates in Rats following Inhalation Exposure (Saillenfait 1999)

Groups of 23 to 25 dams were exposed (6 hours/day, whole-body) to atmospheres containing 2-ethylhexyl acrylate (99.7% purity) at 0, 50, 75, and 100 ppm (approximately 0.375, 0.563, and 0.750 mg/l) during day 6 to day 20 of gestation. From preliminary level-setting studies (no details available) a level of 100 ppm 2-EHA had been reported to provide the highest reliable vapor concentration. Maternal food consumption was measured for the intervals of g.d. 6-13 and of g.d. 13-21. Maternal body weights were recorded on g.d. 0, 6, 13, and 21. Dams were sacrificed on day 21 of gestation and the uteri were removed and weighed. The number of implantation sites, resorptions, and dead and live fetuses were recorded. Uteri, which had no visible implantation sites, were stained with ammonium sulfite (10%) for the detection of early resorptions. At sacrifice live fetuses were weighed, sexed, and examined for external anomalies including those of the oral cavity. Half of live fetuses from each litter were examined for either internal soft tissue or for skeletal changes.

There were no maternal deaths in any of the treatment groups. Dams from the 100-ppm groups showed an absolute weight gain of 24+16 g through the period of exposure, which was lower and statistically significantly different from that of the concurrent control group (42 +/-11 g). Also food intake of 24+3 g food/dam/day through the period of exposure of the 100-ppm group was somewhat lower and statistically significantly different in comparison to that of the concurrent control group (27 +/-2 g food/dam/day). No adverse effects were observed on the mean number of implantation sites per litter and on the mean number of live fetuses per litter in any of the 2-EHA exposed groups. The incidences of non-live implants (3.7-6.4%) and of resorption sites per litter (3.7-6.1%) in the treated groups were lower than those of the concurrent control (both 10.1%).

This observation, however, is not considered to be of toxicological significance. Mean fetal body weights were slightly lower in the treated groups, however not statistically significantly different from that of the concurrent control fetuses. Sex ratio was unaffected. No significant differences were observed between the control and the 2-EHA-treated groups in the incidences of gross anomalies or of visceral or skeletal malformations or variations.

In summary, no embryotoxic, teratogenic or fetotoxic properties of 2-EHA had been revealed from this study for concentrations of up to and including 100 ppm. Due to technical limitations exposure to higher concentrations could not be tested. Based on slightly reduced food intake and lower maternal weight gain at the higher exposure level a NOAEC/maternal toxicity of 75 ppm (approximately 0.563 mg/l) is derived from this study. No embryo-/fetotoxic effects were revealed even at the highest tested concentration at which some signs of maternal toxicity had been observed. Therefore, a NOAEC/developmental toxicity of 100 ppm (approximately 0.750 mg/l) is derived from this study.

In summary, no embryotoxic, teratogenic or fetotoxic properties of 2-EHA had been revealed from this study for concentrations of up to and including 100 ppm. Due to technical limitations exposure to higher concentrations could not be tested. Based on slightly reduced food intake and lower maternal weight gain at the higher exposure level a NOAEC/maternal toxicity of 75 ppm (approximately 0.563 mg/l) is derived from this study. No embryo-/fetotoxic effects were revealed even at the highest tested concentration at which some signs of maternal toxicity had been observed. Therefore, a NOAEC/developmental toxicity of 100 ppm (approximately 0.750 mg/l) is derived from this study.

As described in the section “Toxicokinetics, metabolism and distribution (ADME)” of the dossier, 2-ethylhexyl acrylate is rapidly and extensively absorbed, distributed and eliminated after oral administration. In vitro and in vivo studies have demonstrated that 2-EHA undergo carboxylesterase-catalyzed hydrolysis to acrylic acid and 2-ethylhexanol. The hydrolysis is so fast that exposure to the parent compound is considered to be not significant. Therefore, the developmental toxicity of 2-EHA can be evaluated from studies performed on the substance it-self and its degradation products, acrylic acid (AA) and 2-ethylhexanol (2-EH) and also from a substance, n-butyl acrylate (n-BA), belonging to the same chemical category.

No embryotoxic, teratogenic or fetotoxic properties of 2-EHA had been revealed from an inhalation developmental toxicity study in rats at concentrations of up to and including 100 ppm. The developmental toxicity of 2-EH was evaluated as a part of the CORAP process due to potential concerns on pre-natal development. Nevertheless, it was concluded that this concern could be removed because outcome of a full evaluation of the available information shows that doses of 2-EH are not toxic or are only slightly toxic to maternal animals and no developmental toxicity warranting classification is observed. No prenatal developmental toxicity was observed in rats and rabbits exposed by inhalation to AA, even at concentration that produced some signs of maternal toxicity. No specific teratogenic potential could be revealed for dose levels up to and including 360 ppm (rats) and 225 ppm (rabbits), respectively. After inhalation, n-BA caused fetotoxic effects in rats, but no teratogencity, only at high maternal toxic concentrations (>= 25 ppm). After oral gavage at high doses (>= 1000 mg/kg) to mice, n-BA caused developmental toxicity, only in the presence of maternal toxicity. In an OECD TG #414 study with n-BA in rabbits, a NOAEL of 400 mg/kg bw is derived for both maternal and developmental toxicity based on the absence of adverse effects.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20.10.2021 - 24.01.2023
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Due to unscheduled delays in the contract laboratory, the final report for this study is not yet available, so only the draft report is available so far. However, another dossier update including the final study results and an assessment of its impact on the exposure assessment will be done as soon as possible.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 Jan 2001
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: 11498068E0
- Purity, including information on contaminants, isomers, etc.: 99.8 corr. area-%
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 15-17 weeks
- Weight at study initiation: 3390 - 4513 g
- Fasting period before study: not applicable
- Housing: housed singly in Type 4X03B700CP cages supplied by TECNIPLAST Deutschland GmbH
- Diet (e.g. ad libitum): ad libitum (Kliba maintenance diet rabbit and guinea pig “GLP”, supplied by Granovit AG, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21
- Humidity (%): 46-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light):12/12
Route of administration:
oral: feed
Vehicle:
other: diet
Details on exposure:
Test substance preparation and administration:
The test substance was administered daily as a homogeneous addition to the food with daily concentration adjustment on the basis of body weight and food consumption measurements (GD 6-29).
The test substance preparations were prepared three times: once before the start of the study and two times during the study.
The concentration of 2-Ethylhexyl acrylate in the diet was calculated with the following formula:
Food consumption: 80 g, 130 g or 170 g per day*
Mean body weight: 3900 g*
Depending on the daily individual body weight and daily food consumption of each rabbit, the diet with the most appropriate concentration for the individual animal was chosen each day.
target dose 50 mg/kg bw/d: 1147 ppm (assumed daily food intake of 170 grams); 1500 ppm (assumed daily food intake of 130 grams);2438 ppm (assumed daily food intake of 80 grams)
target dose 150 mg/kg bw/d: 3441/ 4500/ 7313 ppm (assumed daily food intake as specified before)
target dose 300 mg/kg bw/d: 6882/ 9000/ 14625 ppm (assumed daily food intake as specified before)

VEHICLE
Pelleted food (Pelleted maintenance diet rabbit and guinea pig “GLP”); test substance suspensions of appropriate concentrations were prepared and sprayed on the pellets. For the control food, 0.5% Sodium carboxymethyl cellulose suspension in deionized water (with 10 mg/100 mL Cremophor EL) was sprayed on the pellets.
Analytical verification of doses or concentrations:
yes
Remarks:
Samples were sent twice during the study period, for verification of the concentrations, homogeneity and stability.
Details on analytical verification of doses or concentrations:
Samples of the test substance preparations were sent to the analytical laboratory twice during the study period (at the delivery of food and at the end of the administration period) for verification of the concentrations. The samples taken for the first concentration control analysis at the delivery of food were also used to verify the homogeneity of the samples of all concentrations. The concentrations measured at the delivery of food and the end of administration period provided also information on the stability of the substance preparation, as the same preparation was used throughout the entire study period. From each formulation, three samples were taken from the preparation vessel (one from the top, middle and bottom).

Stability analysis
The concentrations measured at the delivery of food and the end of administration period corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations. Therefore, the stability of the test substance preparations over the study period was demonstrated.

Homogeneity analyses of the test substance preparations
The homogeneous distribution of the test substance in the vehicle (pelleted Kliba maintenance diet rabbit and guinea pig “GLP”) was demonstrated in nearly all samples, except samples Nos.6-8 and Nos. 24-26 which did not meet the specification limit of 5%, based on SOP’s of the test facility. This slight deviation from the acceptance criterion was, however, considered acceptable for the analysis of test substances in complex matrices like diet.

Concentration control analyses of the test substance preparations
The results of the analyses of the test substance preparations in pelleted Kliba maintenance diet rabbit and guinea pig “GLP” confirmed the correctness of the prepared concentrations.
The measured concentrations of the samples Nos. 3-5, 6-8, 9-11, 12-14, 15-17, 21-23, 24-26, 27-29, 62-64, 65-67, 68-70, 71-73, 74-76, 77-79, 80-82 and 83-85 corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations (see PART III, Supplement). The mean values for the samples Nos. 18-20 (111.6 %) and 59-61 (86.2 %) of 2-Ethylhexyl acrylate in diet did not meet the specification limit of 90% - 110% of the nominal concentrations. These slight deviations from the acceptance criterion was, however, considered acceptable for the analysis of test substances in complex matrices like diet.
Details on mating procedure:
- Impregnation procedure: artificial insemination
- The day of insemination referred as GD 0 (beginning of the study)of pregnancy
Duration of treatment / exposure:
The test substance was administered to the animals as a homogeneous addition to the food from GD 6-29.
Frequency of treatment:
daily
Duration of test:
30 days
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
191 (225) mg/kg bw/d (effective dose)
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
119 (131) mg/kg bw/d (effective dose)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
39 (42) mg/kg bw/d (effective dose)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group
No. of animals per sex per dose:
25
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: In an initial palatability study, rabbits were exposed to target doses of 100, 300 and 1000 mg/kg bw/d (2294, 6882 and 22941 ppm) 2-Ethylhexyl acrylate in the diet. Compared to the control the average food consumption was reduced by about 21, 24 and 52 % in the low-, mid- and high-dose groups. Based on the extremely reduced food consumption at 1000 mg/kg bw/d resulting in significant weight loss, 500 mg/kg bw/d were chosen as high dose in the subsequent maternal toxicity study.
In the maternal range-finding study rabbits were exposed to target doses of 50, 150 and 500 mg/kg bw/d 2-Ethylhexyl acrylate in the diet. Because of the reduced food consumption, the average substance intake was 45, 136 and 238 mg/kg bw/d in the low-, mid- and high-dose groups. Reduction of food consumption was particularly distinct in the high-dose group and at the beginning of the exposure period, accompanied by a corresponding decrease of body weight. To countervail the circle of ever lower food consumption and higher substance concentration in the diet the animals were kept on the lowest concentration foreseen for the high-dose dose group from GD 10 for the rest of the study. This ensured a relatively stable consumption of the medicated diet but as a consequence the overall substance intake was lower than intended. In the end, the substance intake ranged between 152 and 341 mg/kg bw/d from GD 10 onwards. Two does in the high-dose group aborted, one at mid pregnancy after a long period of food refusal and the other one immediately before term also after a long period of decreased food consumption. Apart from food consumption/body weight effects a slight shift in blood count as well as slightly increased adrenal weights were noted in the high-dose group. Based on these data, the following target doses were chosen for the present prenatal developmental toxicity study in New Zealand White rabbits:
50 mg/kg body weight/day: as low-dose level
150 mg/kg body weight/day: as mid-dose level
300 mg/kg body weight/day: as high-dose level
The intention of the chosen concentrations was to find a balance between a sufficiently high food intake and the achievable dose level.
- Rationale for animal assignment (if not random):
The strain was selected since historical control data is available from the test facility for New Zealand White rabbits. This specific strain has been proven to be sensitive to substances with a teratogenic potential.


Analyses confirmed the correctness of the prepared concentrations, their homogeneous distribution and the stability of the test substance in the vehicle.
The average test substance intake was 39, 119 and 191 mg/kg bw/d, based on all females proven to be pregnant during the treatment period. The reason for the discrepancy to the target doses was reduced food consumption which could not be compensated by applying the procedure described in section 2.2.3. which involved the adaptation of the test substance concentration in the diet to the actual food intake.
However, cases of reduced food consumption were rather irregularly distributed among the individual rabbits within the treatment groups. It is notable, that all females experiencing abortions or complete litter losses had very long time periods of either food refusal or distinctly reduced food consumption, which in turn had a profound influence on average test substance intake of the respective treatment group.
These were the affected animals:
Animal / Day of abortion / Average food consumption (g/animal/day) / Effective dose (test substance intake; mg/kg bw/d)
50 mg/kg bw/d:
27 / GD 26 / 2.5 / 1.0
32 / GD 20 / 2.2 / 0.9
48 / GD 18 / 3.8 / 1.6
150 mg/kg bw/d:
56 / GD 22 / 6.0 / 7.3
66 / - / 2.6 / 2.7
69 / - / 2.9 / 3.3
300 mg/kg bw/d:
76 / GD 27 / 11.7 / 26.9
80 / GD 23 / 5.7 / 7.2
81 / GD 28 / 30.8 / 68.0
83 / GD 28 / 3.7 / 9.3
96 / - / 2.4 / 5.4
Considering the very low effective dose levels in these animals, it is rather unlikely that these abortions/resorptions were caused by the test substance. They were clearly a consequence of the food refusal of these individuals which happened independent of the applied test substance concentrations in the diet, as the relatively equal distribution of the cases about the dose groups shows.
If these outliers were discounted from the calculation of test substance intake, the corresponding group means were about 42 mg/kg bw/d, 131 mg/kg bw/d and 225 mg/kg bw/d for the low-, mid- and high dose group, respectively. Corresponding to similar
values for food consumption, the difference to the target doses in the low- and mid-dose groups.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity (GD 0 through GD 29).

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated based on the obtained results.
- Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- The consumption of food was recorded daily during GD 0-29.
- The intake of test substance was calculated from the amount of food consumed and expressed in milligram per kilogram body weight per day.

POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day 29
- Organs examined:

OTHER:
- Mortality: A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-29).
- Clinical pathology: In the morning blood was taken from the ear vein from not-fasted animals without anesthesia. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
- Pathology:
- Necropsy: After the does have been sacrificed, they were necropsied and assessed by gross pathology. Special attention has been given to the reproductive organs. Animals Nos. 3, 27, 32, 48, 56, 76, 80, 81 and 83 died intercurrently or were sacrificed. They were necropsied and assessed by gross pathology as soon as possible after their death.
- Organ weights: The following weights were determined in all does sacrificed on schedule: 1. Adrenal glands (fixed); 2. Kidneys; 3. Liver; 4. Spleen. All paired organs were weighed together (left and right).
-Organ/tissue fixation: The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution: 1. All gross lesions; 2. Adrenal glands; 3. Kidneys; 4. Liver; 5. Spleen; 6. Stomach. No further examinations or procedures were performed in the study.
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
- Numver of dead fetuses: Yes
After the weight of the uterus had been determined, all subsequent evaluations of the does and the gestational parameters (except of gross pathology and organ weights) were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose, animal numbers were encoded.
Blood sampling:
- Plasma: Yes
- Serum: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter (and the heads of any fetus which revealed severe findings during the external examina-tion, e.g. anophthalmia, microphthalmia or hydrocephalus)
All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.
Statistics:
Statistics of clinical, necropsy and fetal examinations:
DUNNETT-test (twosided) for the hypothesis of equal means:
Food consumptiona), body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each
litter, litter mean fetal body weight, litter mean placental weight

FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions: Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings

WILCOXON-test (one-sided) for the hypothesis of equal medians: Proportions of fetuses with
malformations, variations and/or unclassified observations in each litter

Statistics of clinical pathology:
Blood parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians.

Statistics of pathology:
Organ weights: Non-parametric one-way analysis using the Kruskal-Wallis test (two-sided). If the resulting p-value was equal to or less than 0.05, a pair-wise comparison of each dose group with the control group was performed using the Wilcoxon test (two-sided) for the hypothesis of equal medians.
Carcass weights: Comparison of each group with the control group was performed using the Dunnett test (two-sided) for the hypothesis of equal means.

Indices:
- The conception rate (in %) was calculated according to the following formula:
(number of pregnant animals/ number of fertilized animals) x100
- The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
(number of corpora lutea – number of implantations/ number of corpora lutea) x100
- The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
(number of implantations – number of live fetuses/ number of implantations) x100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
For some does of the test groups 0-3 (0, 50, 150 or 300 mg/kg bw/d), blood in bedding was recorded: control doe No. 9 (GD 23), low-dose does Nos. 26 (GD 26), 32 (GD 19 - this female showed also hypothermia and was sacrificed after abortion on the next day) and 37 (GD 21), mid-dose does Nos. 66 (GD 14, GD 16-18), 69 (GD 12, 13, 17) and high-dose doe No. 96 (GD 17-19). Three females of the mid- and high-dose groups (Nos. 66, 69 and 96) had no viable fetuses.
In total, reduced defecation was observed in five control, 13 low-dose, 13 mid-dose and 19 high-dose females (0, 50, 150 and 300 mg/kg bw/d). No defecation was observed in three control, eight low-dose, five mid-dose and 10 high-dose females. Although frequency of defecation usually shows great variability among individual rabbits across treatment groups during a study, the higher incidence of these observations in the test substance-treated groups comes along with reduced food consumption in the affected animals. There were no further clinical findings in the other does in this study.
Mortality:
mortality observed, treatment-related
Description (incidence):
One female of the control group (No. 3) had to be sacrificed moribund for animal welfare reasons (accidental spinal fracture) on GD 1.
Three low-dose females (50 mg/kg bw/d - Nos. 27 on GD 26, 32 on GD 20, 48 on GD 18), one mid-dose female (150 mg/kg bw/d - No. 56 on GD 22) and four high-dose females (300 mg/kg bw/d - Nos. 76 on GD 27, 80 on GD 23, 81 on GD 28, 83 on GD 28) were sacrificed after abortion ahead of schedule. The affected animals showed a particularly distinct reduction of food consumption during the days upcoming to the abortion.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The high dose (300 mg/kg bw/d) caused a body weight loss in the respective treatment group during the first week of treatment (GD 6-14), followed by a stagnation of body weight during mid pregnancy and some days of recovery shortly before the end of the study. This resulted in statistically significantly reduced body weights on GD 14-25 (up to 8% below control) and some convergence to the control values afterwards.
In the low- and mid-dose groups (50 and 150 mg/kg bw/d) the mean BWC was statistically significantly reduced only at the beginning of treatment (GD 6-9; -32.5 g* [p ≤ 0.05] and -77.4 g** [p ≤ 0.01], respectively) and was normal otherwise considering the high variability of this parameter in rabbits. Body weight was significantly lower in the mid-dose group on a single occasion (GD 16) only.
Although the differences were not statistically significant all treated groups gained less weight in comparison to the concurrent control group during the treatment period (GD 6-29).

Corrected (net) body weight gain:

Mean carcass weight of the does of test group 3 (300 mg/kg bw/d) was reduced (-5%) in comparison to the control group (without attaining statistical significance). Furthermore, the corrected body weight change (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) was impaired in the high-dose group, attaining statistical
significance (-411.1 g*) in comparison to the concurrent control group (-218.1 g).
Mean carcass weights and corrected body weight change of test groups 1 and 2 (50 and 150 mg/kg bw/d) were not significantly different in comparison to the control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In comparison to the control group the mean food consumption of the does in the substance treated groups was reduced during several parts of the treatment period, attaining statistical significance on GD 6-17 (test group 3 - 300 mg/kg bw/d), on GD 6-16 (test group 2 - 150 mg/kg bw/d) and on GD 6-7, GD 9-10 and GD 11-16 (test group 1 - 50 mg/kg bw/d).
However, these average values include all animals who experienced abortions or complete litter loss, too. As these individuals suffered from negligible food intakes over long time periods during the study, and thus skewed the group average considerably, food consumption needed to be evaluated on an individual animal basis.
Intake of test substance:
The amount of test substance (in mg) which was consumed by the animals per kilogram body weight each day was calculated at the times at which food consumption was determined during the administration period (GD 6-29). Test substance intake represents the effective average dose levels which were achieved in the present study.
Mean maternal test substance intake:
Test group 1 (50 mg/kg bw/d): 39.1 mg/kg bw/d
Test group 2 (150 mg/kg bw/d): 118.8 mg/kg bw/d
Test group 3 (300 mg/kg bw/d): 191.3 mg/kg bw/d
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
At study day 29, in does of test group 1 (50 mg/kg bw/d) absolute and relative neutrophil counts were significantly decreased whereas absolute and relative monocyte counts, and relative lymphocyte counts were significantly increased. Additionally, in does of test group 2 (150 mg/kg bw/d) absolute neutrophil counts were significantly decreased whereas relative monocyte counts were significantly increased. All mentioned alterations were not dose dependent.
Therefore, these changes were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
At study day 29, in does of test group 1 (50 mg/kg bw/d) glucose levels were significantly increased, but the change was not dose dependent. Therefore, this alteration was regarded as incidental and not treatment-related.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The mean gravid uterus weights of the rabbits of test groups 1-3 (50, 150 or 300 mg/kg bw/d) were not significantly different from controls. The differences between these groups and the control group showed no dose-dependency and were assessed to be without biological relevance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and
treatment groups. They were considered to be incidental or spontaneous in origin and without
any relation to treatment.
Details on results:
Clinical examinations
Only pregnant does were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant does with scheduled sacrifice (GD 29) were used for the calculation of mean gravid uterine weights, mean organ weights, corrected (net) body weight gain and summary of reproduction data.
The following females were excluded from the above-mentioned calculations:
Test group 0 (0 mg/kg bw/d):
• females Nos. 2, 4, 6, 24 - not pregnant
• female No. 3 - sacrificed moribund
Test group 1 (50 mg/kg bw/d):
• females Nos. 29, 30, 40 - not pregnant
• females Nos. 27, 32, 48 - sacrificed after abortion
Test group 2 (150 mg/kg bw/d):
• female No. 63 - not pregnant
• female No. 56 - sacrificed after abortion
Test group 3 (300 mg/kg bw/d):
• females Nos. 78, 97 - not pregnant
• females Nos. 76, 80, 81, 83 - sacrificed after abortion
Thus, according to the requirements of the corresponding test guidelines, each test group including the controls contained a sufficient number of females with implantation sites at necropsy (approximately 20, but not fewer than 16 females with implantation sites).

- Absolute weights: All mean absolute weight parameters did not show significant differences when compared to the control group 0.
- Relative organ weights: All mean relative weight parameters did not show significant differences when compared to the control group 0.
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
Resorption rate (i.e. post-implantation loss) was not statistically significantly different from control in any of the treatment groups, however the mid- and high-dose values (17.0 and 16.4 mean%) were above the historical range of the test facility (2.1 – 11.3 mean%). This was due to two females of the mid-dose group (Nos. 66 and 69) and one female of the high-dose group (No. 96) which had no viable fetuses but just early resorptions in their uteri. The resulting 100% post-implantation loss in these animals contributed higher than average to the group means.
These litter losses were associated with extensive periods of food refusal by these animals and were regarded as not being related to a potential developmental toxicity of the test compound.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Two dead fetuses from high-dose doe No. 77 are within the historical control range and therefore considered to be an incidental finding.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
conception rates were 84% / 88% / 96% / 92% in test groups 0-3 (0, 50, 150 and 300 mg/kg bw/d)
Details on maternal toxic effects:
There were no test substance-related and/or biologically relevant differences between the test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre-implantation loss and viable fetuses. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.
Key result
Dose descriptor:
NOAEL
Effect level:
191 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: high dose
Remarks on result:
other: prenatal developmental toxicity
Dose descriptor:
NOAEL
Effect level:
119 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
food consumption and compound intake
Remarks on result:
other: maternal toxicity
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean weight of the male high-dose fetuses (300 mg/kg bw/d) was slightly lower than concurrent control (-12%). Although the difference attained statistical significance, the mean male fetal weight was well within range of 95% spread of the historical control data (HCD: mean 37.1 g [24.8 - 49.4]). The mean weight of the high-dose female fetuses was comparable to the concurrent control. When both sexes were combined, the mean fetal weight of the high-dose group was also comparable to control. The mean group value (35.6 g) was close to the mean and well in the range of 95% spread of the historical control data (HCD: mean 36.8 [24.4 -49.2]). The slightly lower average weight of the high-dose fetuses can be attributed to the larger average litter size in this group compared to the control, the same effect is also noticeable in the low-dose group.
The mean fetal weights of test groups 1 and 2 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (50, 150 and 300 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- One external malformation (‘umbilical hernia’) was recorded for two control fetuses (0 mg/kg bw/d). Female fetus No. 55-01 (150 mg/kg bw/d) had multiple external malformations concerning its whole fetal body (compacted appearance, multiple bulges in the region of the neck, throat and axilla of both forelimbs) (see Attachments, Table 1).
-The distribution of external malformations about the dose groups does not indicate an association to the tre atment, no statistically significant differences between the groups were noted (see Attachments, Table 2).
- No external variations were recorded.
- One unclassified external observation was recorded. ‘Placentae necrobiotic’ was seen in one fetus of the control group and is considered as incidental (see Attachments, Table 3).
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- One skeletal malformation was recorded for a single mid-dose fetus (150 mg/kg bw/d). This finding is considered to be spontaneous in origin and not treatment-related (see Attachments, Table 8 + Table 9).
Fetal skeletal variations:
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dosing. The addition of skeletal variations resulted in an increased affected fetuses per litter incidence in test group 2, attaining statistical significance. However, due to the lack of dose-response relationship and the fact, that the overall incidence of test group 2 was only slightly above the historical control range, while the other incidences of test groups 0, 1 and 3 were well within (HCD: mean% 91.7 [84.9 - 97.2]), this increase is evaluated to be spontaneous in origin and not treatment-related (see Attachments, Table 10).
Fetal skeletal unclassified cartilage observations:
Some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the sternum and the ribs and did not show any relation to dosing. Therefore, they are assessed as not treatment-related (see Attachments, Table 11).
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Soft tissue malformations occurred in all test groups including controls (0, 50, 150 or 300 mg/kg bw/d). All listed findings are part of the normal background of malformations in this rabbit strain as they are all present in the historical control data. In terms of incidences no statistically significant nor toxicologically relevant differences between the groups were noted, and the overall malformation rate was inside the historical control range (see Attachments, Table 4 + Table 5).
Details on embryotoxic / teratogenic effects:
Fetal soft tissue variations:
The examinations of the soft tissues revealed cystic dilatation of the brain, dilated aorta, malpositioned carotid branch, absent lung lobe (Lobus inferior medialis) and dilated renal pelvis in individual fetuses of the different test groups (0, 50, 150 or 300 mg/kg bw/d). No statistically significant or toxicologically relevant differences between the groups were noted, and the overall incidences were within the historical control range (see Attachments, Table 6).

Fetal soft tissue unclassified observations:
Two unclassified soft tissue observations were recorded: discolored thymus in two mid-dose fetuses and a blood coagulum around urinary bladder in fetuses of all test groups. These findings are considered to be incidental (see Attachments, Table 7).

Assessment of all fetal external, soft tissue and skeletal observations:
There were noted external, soft tissue and skeletal malformations in all substance-treated test groups (50, 150 or 300 mg/kg bw/d) as well as in the control. The distribution of total malformations about the groups was not related to dose, in fact the lowest rate was noted in the high-dose group.

 In total, five fetuses had more than one malformation. Female control fetus No. 7-06, low-dose male fetus No. 49-06 (50 mg/kg bw/d) and high-dose female fetus No. 89-05 (300 mg/kg bw/d) had malpositioned kidneys combined with short ureter, respectively. For low-dose male fetus No. 49-04 an absent subclavian, malpositioned kidney and a short ureter were recorded. Furthermore, female mid-dose fetus No. 55-01 (150 mg/kg bw/d) had multiple external
malformations concerning its whole fetal body (compacted appearance, multiple bulges in the region of the neck, throat and axilla of both forelimbs). No ontogenetic pattern is recognizable for the individual malformations nor was there a cluster of any of these individual malformations seen in the other offspring of these test groups.
Further malformations, such as umbilical hernia (test group 0), enlarged lens (test groups 1-3) and severely fused sternebrae (bony plate) (test group 2) were scattered observations in individual fetuses. All these findings are not dose-related and part of the normal background of malformations in this rabbit strain as they are all present in the historical control data. Thus, a relation to the treatment is not assumed (see Attachments, Table 12).

External variations did not occur in any fetus in this study. A spontaneous origin is assumed for the soft tissue variations and the broad range of skeletal variations which were observed in fetuses of all test groups including the controls (see Attachments, Table 13).
Although the total rate of affected fetuses per litter was statistically significantly increased in the mid-dose group (150 mg/kg bw/d), the overall incidences of all classified fetal variations were comparable to the historical control range (HCD: mean% 91.98 [86.02 - 97.16]). Due to the lack of dose-response relationship and the fact, that the overall incidence of test group 2 was only slightly above the historical control range, the increase is considered to be spontaneous in origin and not treatment-related.

A spontaneous origin is assumed for the unclassified external, the unclassified soft tissue and the unclassified skeletal cartilage observations, which were observed in several fetuses of test groups 0-3. The distribution and type of these findings do not suggest any relation to treatment. Thus, fetal examinations revealed that there is no adverse effect of the compound on the respective morphological structures up to the highest dose tested (300 mg/kg bw/d).
Dose descriptor:
NOAEL
Effect level:
191 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: high dose
Developmental effects observed:
no
Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of 2-Ethylhexyl acrylate as homogeneous inclusion in the diet to pregnant New Zealand White rabbits from implantation to the expected day of parturition (GD 6-29) caused evidence indicative of maternal toxicity (food refusal because of stomach irritation) at the highest dose. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is effectively 119 mg/kg bw/d.
Since there was no evidence for treatment-related adverse effects of the test substance on fetal morphology the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is effectively 191 mg/kg bw/d.

The test substance is considered not teratogenic in rabbits under the test conditions employed in this study.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication, comparable to current guidelines
Principles of method if other than guideline:
Groups of 20-29 bred female rats (17-25 pregnant) were exposed to the compound 6h/day on days 6 through 20 of gestation by inhalation. Control animals were exposed concurrently to filtered room air. The test concentrations of ethylhexyl acrylate were 50, 75, and 100 ppm (corresponding to approx. 0.38, 0.56, and 0.75 mg/L)*.
* Calculation of concentrations (mg/L) based on Derelanko MJ (2000). Toxicologist's Pocket Handbook, CRC Press, conversion table, p. 57
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: IFFA CREDO Breeding Laboratories (Saint-Germain-sur-l' Arbresle, France)
- Age at study initiation: Young, nulliparous females
- Weight at study initiation: 200-220 g
- Housing: Single in clear polycarbonate cages with stainless-steel wire lids and hardwood shaving as bedding.
- Diet: Food pellets (UAR Alimentation Villemoisson, France), ad libitum
- Water: Filtered tap water, ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 5
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
EXPOSURE
Exposures were conducted in 200-L glass/stainless-steel inhalation chambers with dynamic and adjustable laminar air flow (6-20 m3/h). The chamber temperature was set at 23 ± 2°C, and the relative humidity at 50 ± 5 %. The air-flow rate passed through the fritted disk of a heated bubbler containing the test chemical. Concentrations of acrylate ester were monitored continuously with a gas-chromatograph equipped with a flame ionization detector and an automatic gas-sampling valve. In addition, exposure levels were determined once during each 6-h exposure period by collecting atmosphere samples through glass tubes packed with activated charcoal. The charcoal samples were then desorbed with carbon disulfide. The resulting samples were analyzed by gas chromatography using appropriate internal standards. Since 2-EHA has a rather low vapour pressure (0.14 mm Hg at 20 °C), the presence of liquid particles was evaluated at the highest concentration generated (i.e. 100 ppm). Airborn particles were measured with an Aerodynamic Particle Sizer.
No differences in particle counts were observed between the clean filtered air (control) and the vapor-laden air in the exposure chambers.


GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Glass/stainless-steel inhalation chambers
- Source and rate of air: Test atmospheres were generated through an additional air-flow  rate passed through the fritted disk of a heated bubbler containing ethylhexyl acrylate.  The vaporized compound was introduced into the main air inlet pipe of the exposure chamber.
- Air flow rate: 6-20 m3/h


TEST ATMOSPHERE
- Brief description of analytical method used: GC/FID
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical concentrations (mean ± SD):
51.0 ± 4.3 ppm (nominal: 50 ppm)
75.4 ± 9.2 ppm (nominal: 75 ppm)
102.5 ± 7.9 ppm (nominal: 100 ppm)
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2-3
- Length of cohabitation: Overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
day 6-20 of gestation
Frequency of treatment:
6h /day
Duration of test:
until gestation day 21
Dose / conc.:
50 ppm
Remarks:
0.375 mg/L
Dose / conc.:
75 ppm
Remarks:
0.563 mg/L
Dose / conc.:
100 ppm
Remarks:
0.750 mg/L
No. of animals per sex per dose:
20
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale:
Exposure concentrations were based on preliminary studies. Only minimal maternal toxicity was observed after exposure to 90 ppm ethylhexyl acrylate. Nevertheless, l00 ppm ethylhexyl acrylate was used as the highest concentration for the definitive developmental toxicity study, since preliminary level-setting studies have indicated that 100 ppm was the highest reliable vapor concentration technically possible.

Maternal examinations:
BODY WEIGHT: Yes
- Time schedule for examinations: On gestation day (GD) 0, 6, 13 and 21.


FOOD CONSUMPTION: YES
Food consumption was measured for the intervals GD 6-13 and 13-21.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: The uteri were removed and weighed. The number of implantation sites, resorptions, and dead and live fetuses were recorded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
The number of implantation sites, resorptions, and dead and live fetuses were recorded. Uteri which had no visible implantation sites were stained with ammonium sulfide (10 %) to detect very early resorptions.

Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
Live fetuses were weighed, sexed, and examined for external anomalies including those of the oral cavity. Half of the live fetuses from each litter were preserved in Bouin's solution and examined for internal soft tissue changes. The other half were fixed in ethanol (70 %), eviscerated, and then processed for skeletal staining with alizarin red S for subsequent skeletal examination.

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter
Statistics:
Data were presented as mean ± SD. The number of  implantation sites and live fetuses and the various body weights were analyzed by one-way analysis of variance (ANOVA), followed by Dunnett's test if differences were found. The percentages of non-live  implants and  resorptions and the proportions of fetuses with alterations in each litter were evaluated by using the Kruskal-Wallis test, followed by the Dixon-Massey test where appropriate. Rates of pregnancy, fetal sex ratio, and percentage of litters with malformations or external, visceral, or skeletal variations were analyzed by using Fisher's test. Where applicable, least-squares analysis was carried out. For all statistical tests, the level of significance was set a priori at alpha = 0.05.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No test dams died. Except for a significant decrease in absolute weight gain at 100 ppm, there were no significant changes in maternal weight gain of females exposed to ethylhexyl acrylate, compared of those of controls. Rats from the 100 ppm-group showed a significant decrease in food intake throughout the entire exposure period (8-11 % reduction).
Dose descriptor:
NOAEC
Effect level:
75 ppm
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Abnormalities:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No adverse effects were observed on the mean number of implantations and live fetuses among litters exposed to ethylhexyl acrylate. The statistically significant reductions in the incidence of non-live implants and resorptions at 50 and 100 ppm were not considered to be of toxicological significance. There was a statistically significant trend toward decreased fetal body weights, but the pairwise comparisons to the concurrent control group were not significantly different. No significant differences were observed between control and treated groups in the incidence of fetal malformations or variations.
Dose descriptor:
NOAEC
Effect level:
100 ppm
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Developmental effects observed:
no

Maternal body weights:

Concentration [ppm/6h/d]

Maternal body weight GD 6 [g]

Absolute weight gain [g]

0

289 ± 17

42 ± 11

50

294 ± 19

35 ± 15

75

299 ± 21

32 ± 18

100

292 ± 18

24 ± 16**

Absolute weight gain: (Day 21 body weight) - (gravid uterus weight) - (Day 6 body weight)

Reproductive parameters:

 

Conc. [ppm/6h/d]

No. of litters

No. of implantation sites/litter

% of non-live implants/litter

% of resorption sites/litter

No. of live fetuses/litter

Average fetal body weight/litter [g]

0

25

15.64 ± 2.87

10.12 ± 10.57

10.12±10.57

14.20 ± 3.57

 5.74 ± 0.33

50

24

15.42 ± 4.23

3.65 ± 5.26*

3.65 ± 5.26*

14.88 ± 4.21

 5.69 ± 0.41

75

23

16.30 ± 3.80

6.44 ± 8.26

6.07 ± 7.97

15.26 ± 3.99

 5.58 ± 0.40

100

23

15.96 ± 2.46

3.93 ± 4.54*

3.93 ± 4.54*

15.30 ± 2.27

 5.53 ± 0.31

Concentration [ppm/6h/d]

0

50

75

100

Mean % of fetuses with:

- any malformations/litter

0.27 ± 1.33

1.89 ± 6.91

0.94 ± 2.54

0

- external variations/litter

0

4.41 ± 20.39

0

0

- visceral variations/litter

2.46 ± 5.98

7.33 ± 14.90

8.27 ± 11.36

4.76 ± 10.54

- skeletal variations/litter

20.62 ± 20.98

22.34 ± 18.63

17.40 ± 21.21

16.30 ± 15.21

- any variations/litter

11.86 ± 10.96

19.09 ± 21.09

12.78 ± 11.49

10.62 ± 10.63

* ,** Significant differences from the control (0 ppm) value, p 0.05, and p 0.01, respectively.

Conclusions:
In summary, no embryotoxic, teratogenic or fetotoxic properties of 2-EHA had been revealed from this study for concentrations of up to and including 100 ppm. Due to technical limitations exposure to higher concentrations could not be tested. Based on slightly reduced food intake and lower maternal weight gain at the higher exposure level a NOAEC/maternal toxicity of 75 ppm (approximately 0.563 mg/l) is derived from this study. No embryo-/fetotoxic effects were revealed even at the highest tested concentration at which some signs of maternal toxicity had been observed. Therefore, a NOAEC/developmental toxicity of 100 ppm (approximately 0.750 mg/l) is derived from this study.
Executive summary:

Groups of 23 to 25 dams were exposed (6 hours/day, whole-body) to atmospheres containing 2-ethylhexyl acrylate (99.7% purity) at 0, 50, 75, and 100 ppm (approximately 0.375, 0.563, and 0.750 mg/l) during day 6 to day 20 of gestation. From preliminary level-setting studies (no details available) a level of 100 ppm 2-EHA had been reported to provide the highest reliable vapor concentration. Maternal food consumption was measured for the intervals of g.d. 6-13 and of g.d. 13-21. Maternal body weights were recorded on g.d. 0, 6, 13, and 21. Dams were sacrificed on day 21 of gestation and the uteri were removed and weighed. The number of implantation sites, resorptions, and dead and live fetuses were recorded. Uteri, which had no visible implantation sites, were stained with ammonium sulfite (10%) for the detection of early resorptions. At sacrifice live fetuses were weighed, sexed, and examined for external anomalies including those of the oral cavity. Half of live fetuses from each litter were examined for either internal soft tissue or for skeletal changes.

There were no maternal deaths in any of the treatment groups. Dams from the 100-ppm groups showed an absolute weight gain of 24+16 g through the period of exposure, which was lower and statistically significantly different from that of the concurrent control group (42 +/-11 g). Also food intake of 24+3 g food/dam/day through the period of exposure of the 100-ppm group was somewhat lower and statistically significantly different in comparison to that of the concurrent control group (27 +/-2 g food/dam/day). No adverse effects were observed on the mean number of implantation sites per litter and on the mean number of live fetuses per litter in any of the 2-EHA exposed groups. The incidences of non-live implants (3.7-6.4%) and of resorption sites per litter (3.7-6.1%) in the treated groups were lower than those of the concurrent control (both 10.1%).

This observation, however, is not considered to be of toxicological significance. Mean fetal body weights were slightly lower in the treated groups, however not statistically significantly different from that of the concurrent control fetuses. Sex ratio was unaffected. No significant differences were observed between the control and the 2-EHA-treated groups in the incidences of gross anomalies or of visceral or skeletal malformations or variations.

In summary, no embryotoxic, teratogenic or fetotoxic properties of 2-EHA had been revealed from this study for concentrations of up to and including 100 ppm. Due to technical limitations exposure to higher concentrations could not be tested. Based on slightly reduced food intake and lower maternal weight gain at the higher exposure level a NOAEC/maternal toxicity of 75 ppm (approximately 0.563 mg/l) is derived from this study. No embryo-/fetotoxic effects were revealed even at the highest tested concentration at which some signs of maternal toxicity had been observed. Therefore, a NOAEC/developmental toxicity of 100 ppm (approximately 0.750 mg/l) is derived from this study.

Endpoint:
developmental toxicity
Type of information:
other: Corap assessment
Adequacy of study:
weight of evidence
Reliability:
other: Substance evaluation under CoRAP
Principles of method if other than guideline:
Principles of method if other than guideline
2-Ethylhexan-1-ol was originally selected for substance evaluation in order to clarify suspected risks
about:
- CMR properties, in particular developmental toxicity
Species:
other: rodent and non-rodent
Dose descriptor:
other: Corap evaluation
Remarks on result:
other: 2-EH is not a developmental toxicant
Abnormalities:
no effects observed
Dose descriptor:
other: Corap evaluation
Remarks on result:
other: 2-EH is not a developmental toxicant
Developmental effects observed:
no
Conclusions:
The effects on the reproduction of 2-EH were evaluated in CoRAP in 2015:
•       The results of animal studies provide evidence of an adverse effect of 2-EH on development at very high doses [> 1000 mg/kg bw/d] causing strong toxic effects in dams, therefore they can be considered as a secondary non-specific consequence of other toxic effects. Evaluation of the available information shows that no maternal toxicity or slight maternal toxicity was observed with in animal studies and no developmental toxicity warranting classification is observed.
It was concluded that this concern could be removed because outcome of a full evaluation of the available information shows that doses of 2-EH are not toxic or are only slightly toxic to maternal animals and no developmental toxicity warranting classification is observed.
Executive summary:

Excerpt from the CoRAP evaluation report:

Toxicity to reproduction (effects on fertility and developmental toxicity)

 

One of the reasons for the selection of 2-EH was health hazard concern. It was noted in the screening that developmental effects were observed in the pre-natal developmental toxicity studies. Therefore, a full evaluation of the available information was required in order to assess whether the observed developmental effects are the result of maternal toxicity. This part of the document reviews the available study reports in order to assess reproductive toxicity of 2-EH, and assess whether a proposal of harmonised C&L is needed for this endpoint.

Developmental toxicit

 

There are several studies to evaluate developmental toxicity of 2-EH: two studies in mice (study report 1991; Hardin et al. 1987) and four studies in rats (Hellwig and Jäckh, 1997; Ritter et al. 1987, Tyl et al. 1992, Nelson et al. 1989).

 

Mice

 

The first one, GLP and OECD TG 414 compliant study (study report 1991 reported also as NTP, 1991) provided evidence that 2-EH in doses 17, 59, and 191 mg/kg bw/day does not induce embryo or fetal toxicity in mice.

 

In the second mice study (Hardin et al. 1987) in which mice were given 1525 mg/kg on day 6 till day 13 of gestation the following developmental effects were observed: decreased number of viable litters and pups per liter, decreased birth weight and weight gain for pups. However these effects should be considered as a secondary non-specific consequence of other toxic effects in dams, because 2-EH at a dose applied in this study caused 34% mortality in the exposed mice. In addition, short term toxicity study in mice using similar dose of 1500 mg/kg/d for (study report, 1992b) provided evidence that 2- EH at this repeated dose level causes damage of several organs (stomach, liver, kidney) which may affect intrauterine and early postnatal development of pups.

 

Rats

 

There were two oral studies (Hellwig and Jäckh, 1997; Ritter et al. 1987), one dermal study ( Tyl et al. 1992) and one inhalation study (Neslon et al. 1989) of developmental toxicity of 2-EH in rats.

 

In the Hellwig and Jäckh oral study (1997) performed in accordance with GLP and OECD TG 414 (except that 10 animals instead of 20 was used per group) 2-EH at dose of 1300 mg/kg/d caused intrauterine deaths of embryos and pups, reduced foetal weight, increased incidence of internal and skeletal malformations as well as of skeletal variations and retardation. However, 2-EH at the dose of 1300 mg/kg/d was also very toxic to dams; 6 out of 10 treated dams were found dead before the end of the study. Therefore high maternal toxicity was most probably responsible for some effects such as increased intrauterine deaths or reduced foetal weight, as well as for some of the internal and skeletal malformations observed in that group, although their incidence was very low. 2-EH did not induced developmental toxicity at a dose of 130 mg/kg/d, while at a dose of 650 mg/kg/d the foetal weight slightly reduced, but still within historical control level. Therefore it may be concluded that in this study 2-EH did not exert developmental toxicity in doses not lethal to dams.

 

The oral study of Ritter et al. (1987) has limited reliability because the study design was not similar to that required by OECD TG 414 or method B.31 (Council Regulation (EC) No 440/2008). In addition, the observed results were not consistent with those observed in other studies. In spite of high doses used (800 and 1600 mg/kg/d) the study did not provide any information on maternal toxicity and no embryo or pup mortality was observed. Malformations in fetuses following single treatment with 2-EH at a dose approximately 1600 mg/kg included hydronephrosis (7.8% of live fetuses), tail defects (4.9% of live fetuses), limb defects (9.7% of live fetuses). Such defects were not observed after 2-EH treatment in studies of Hellwig and Jäckh (1997) and study report (1991) or in other studies. Taking into account a purpose of the study focused on clarification of developmental toxicity of di(2-ethylhexyl) phthalate (DEHP, use of only 7 females in a group, application of 2-EH only as a single dose at 12 day of gestation, lack of compliance of study design with OECD TG 414, lack of consistency of effects observed with other studies, it is doubtful whether study of Ritter et al. (1987) should be taken as a reliable source of information on developmental toxicity of 2-ethylhexanol.

 

The embryonic or fetal development of rats was not affected in the dermal developmental toxicity study (Tyl et al., 1992) conducted under GLP and with a design compliant with OECD TG 414 in which 2-EH was administered at doses 252, 840, and 2520 mg/kg bw/day, although the systemic maternal toxicity was observed at the highest dose.

 

No developmental toxicity was noted in an inhalation study (Nelson et al., 1989), in which female rats were exposed 2-EH at 850 mg/m³ during days 0-19 of gestation. At this exposure level 2-EH was moderately toxic to dams as can be judged based on reduction of feed consumption and body weight gain during gestation.

 

Based on the existing body of evidence it is concluded that at doses not lethal to mothers, in studies performed in compliance with methodological requirements, 2-EH does not induce developmental toxicity in mice and rats. Only at high doses, which were lethal to dams 2-EH increases intrauterine lethality of embryos and pups and leads to retardation of development. Even at these lethal doses the increase of fetal malformations is very low and no dose-response relationship is seen.

 

It is concluded that these developmental effects at doses highly toxic to dams are secondary non-specific consequence of other toxic effects in dams, and they do not justify classification of 2-EH for developmental toxicity. Several studies (study report 1991 reported also as NTP, 1991; Hellwig and Jäckh, 1997; Tyl et al., 1992 and Nelson et al., 1989) provided evidence that 2-EH is not a developmental toxicant in rats and mice.

 

2-ethylhexanoic acid (2-EHA)

 

The substance evaluation for 2-EHA is currently ongoing and the conclusions of this evaluation will be published on ECHA website once the evaluation is concluded.

 

2-ethylhexanoic acid (2-EHA) (EC No205-743-6, CAS No 149-57-5) ), which is major urinary metabolite of 2-ethyl-1-hexanol in rats (Albro, 1975; Deisinger et al. 1993;1994) has harmonised classification as Repr. Cat. 3; R63 Possible risk of harm to the unborn child, which has been transposed to Repr. 2 H361d *** in Table 3.1 List of harmonised classification and labelling of hazardous substances of Annex VI of the Regulation 1272/2008. In the study by Deisinger et al. (1994), the main metabolites in urine of orally treated rats were 2-ethylhexanoic acid, 5-hydroxy-2-ethylhexanoic acid, 6- hydroxy-2-ethylhexanoic acid and 2-ethyl-1,6-hexane diacid. Together, they represented 37 - 45% of the administered dose. Minor metabolites were 5-hydroxy-2-ethylhexanoic acid as well as lactones of 5-hydroxy-2-ethylhexanoic acid and 2-ethyl-5-hexanoneacid. They represented 3 - 5% of the administered dose. About 1% of the administered dose was recovered as 2-ethylhexanol. All these compounds were predominantly excreted as glucuronides (Deisinger et al.,1994). Albro (1975) reported the formation of about 50% 2-ethylhexanoic acid following a single oral exposure of rats to 275 mg/kg.

 

1.  The range-finding study in Fischer 344 rats showed significant maternal toxicity (death in seven of eight dams) and statistically significant reduction of maternal weight gain at 1000 mg/kg for GD 6-9. Indications of maternal toxicity were also observed at 500 mg/kg, including not statistically significant weight gain reduction and clinical sings of toxicity.

 

In the main developmental toxicity study, groups of 25 pregnant Fischer 344 rats per dose level received daily doses of 0, 100, 250 and 500 mg/kg 2-EHA (nominal in corn oil) by oral gavage from gestational day 6 to 15 (study report, 1988c; 1988d; study report, 1993 ) the clinical signs of maternal toxicity were only observed at the high-dose level (500 mg/kg) and included hypoactivity, ataxia, audible respiration, ocular discharge and periocular encrustations. No mortality and no effects on body weight were observed. Liver weight (absolute and relative) was significantly increased in the high-dose group.

 

Foetal effects: There were no changes in the incidence of resorptions and dead foetuses or in the percentage of viable foetuses. Foetal body weights (males and females) per litter were significantly reduced at 500 mg/kg, but these findings may be confounded by the slightly larger mean litter size. No significant differences in the incidence of external, skeletal or visceral malformations were observed among all groups.

 

There was a reduction in ossification of the axial and appendicular skeletons at 500 mg/kg. An increase in the number of foetuses with unossified anterior arch of the atlas and proximal phalanges of the forelimb and hindlimb was also observed at 250 mg/kg.

NOAEL of 250 mg/kg for maternal toxicity of 2-EHA was obtained, based on clinical signs of toxicity and increased liver weights. For developmental toxicity, a NOAEL of 100 mg/kg was established, based on reduced skeletal ossification at a dose of 250mg/kg/d. (study report, 1988c; 1988d; study report, 1993). However, minor developmental changes, when there is only a small reduction in foetal/pup body weight or retardation of ossification when seen in association with maternal toxicity, do not necessarily warrant classification as developmental toxicant (point 3.7.2.4.3. of the CLP Regulation).

2. The range-finding study in pregnant rabbits treated with 500 and 1000 mg/kg 2-EHA showed high toxicity. Mortality was observed at the high and mid doses. No changes in resorptions, deaths or malformed foetuses occurred. No external malformations were observed in foetuses at any of the treated groups (study report, 1988c; 1988d; study report, 1993).

In the definitive study, groups of 15 pregnant rabbits per dose level were administered, by gavage, daily doses of 0, 25, 125 and 250 mg/kg 2-EHA in corn oil on gestational days 6 to 18. In this study, mortality was recorded at 125 and 250 mg/kg (one female each) on days 15 and 16 of gestation, respectively. One abortion was observed on gestational day 27 at 125 mg/kg. A significant reduction in body weight gain and food consumption was observed in the high-dose group during the post-treatment period (gestational days 18 to 29). At necropsy, no gross pathology, no changes in corrected body or gestational weights or in absolute and relative liver weights were observed.

 

Foetal effects: There was no increase of resorptions and dead foetuses or changes in the percentage of viable foetuses. No effects on foetal body weights and sex ratios were observed and no differences in malformations or variations were seen either.

3.  In addition, a non-GLP developmental toxicity study, equivalent or similar to OECD 414, has been reported in the IUCLID dataset and considered as the supporting study (Pennanenet al., 1992). Groups of 20 or 21 female Wistar rats per dose level received daily doses of 100, 300 and 600 mg/kg 2-EHA as sodium salt via drinking water, during gestational days 6 to 19. Control animals received deionized water.

 

Body weight of dams suffered a slight decrease at the high-dose level from day 13 onward. At termination, statistically significant reductions in mean body weight and corrected maternal body weight gain were observed. In the same dose group, a decrease of 20% in the consumption of drinking water containing 2-EHA was seen from day 6, compared to the control group. No differences in food consumption were observed at any dose level. No maternal toxicity was noted at the low- and mid-dose groups.

In the mid- and high-dose groups the placental weight was also statistically significant reduced. No changes in gravid uterus weight were observed. At necropsy, no gross pathological changes in the organs of the dams occurred.

 

Foetal effects: The number of implantations, living foetuses or resorptions were not affected by treatment with 2-EHA . No dead foetuses were seen either in treated or control groups. Significant decreases in mean foetal body weight per litter were observed at 600 mg/kg. At 300 mg/kg, the mean body weight of female foetuses was also decreased.

 

Dose-dependent increases in the number of foetuses with skeletal or visceral anomalies were observed at all dose levels, compared to controls. It has to be pointed out that the number of litters affected by these alterations has not been indicated. Clubfoot (congenital deformity of the foot, which is twisted out of shape or position) was the most severe skeletal malformation, occurred in all treatment groups, being only statistically significant at the two highest doses.

Those results justified the EU harmonised classification of 2-ethylhexanoic acid as Repr. 2 H361d: Suspected of damaging the unborn child based on the observed developmental effects in animals, such as fetotoxicity and skeletal malformations (clubfoot) in rat following oral doses given on days 6-19 of gestation.

 

The analysis of data on of 2-EHA indicate that potency of its developmental toxicity in rats is rather low, while it does not exert developmental toxicity in rabbits, since no embryonal or foetal effects were observed in rabbits given 2-EHA et doses of 25, 125, 250, 500 and 1000 mg/kg (study report, 1993). In rats the developmental toxicity is shown as retardation in ossification of skeletal system in fetuses of rats exposed at doses of 250 and 500 mg/kg/d. (study report (1993)) or as a decreased foetal body weight, and increases incidence skeletal or visceral anomalies and of clubfoot in foetuses of females rats exposed to EHA at 300 and 600 mg/kg.

 

Assuming that in the worst case 50 % of orally given 2-EH is metabolised to 2-EHA in order to reach developmental toxicity the doses of 2-EH would have to be at the level of 6001200 mg/kg, which are known to be rather highly toxic to adult rats (Hellwig and Jäckh, 1997; study report, 1991). Therefore the data showing developmental toxicity of 2-EHA do not provide sufficient evidence of developmental toxicity of the parent substance.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in compliance with GLP regulations.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Wiga GmbH, D-Sulzfeld
- Age at study initiation: 10 weeks old
- Weight at study initiation: mean weight 215 g
- Housing: Single in Makrolon/wire cages (type MD III supplied by Becker & Co., Castrop-Rauxel)
- Diet (ad libitum): SSNIFF R meal, Ssniff Versuchstierdiaeten GmbH, CH-Soest
- Water (ad libitum): Tap water


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 5
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
Exposures were conducted using a continuous infusion pump (type Infu 362). Acrylic acid was metered into the inner coil of a glass evaporator heated by hot water. The acrylic acid vapours were diluted with a flow of fresh conditioned air with a flow meter. This mixture of acrylic acid vapour and air was passed through a glass cooler, thermostated cold water at 8-10°C being passed through its inner coil. The mixture of acrylic acid vapour and air was thus cooled to 23°C and passed to the exposure chambers (made of glass and steel, volume about 500 liters). The following amounts of test substance were used: 1.15 ml/h (study group 1, 40 ppm), 3.4 ml/h (study group 2, 120 ppm), 10.2 ml/h (study group 3, 360 ppm). The temperature in all exposure chambers was continuously monitored using NTC sensors and the measured values were recorded using a 12-channel printer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of acrylic acid in the exposure chambers was determined using a continuously operating total hydrocarbons analyzer equipped with a flame ionization detector.
Nominal concentrations (ppm): 40.7, 120.3, 361
Analytical concentrations (ppm): 39.4, 114.0, 356.2
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: 15.5 hours
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
from day 6 to day 15 of gestation
Frequency of treatment:
6 hours/day
Duration of test:
until day 20 of gestation
Dose / conc.:
0.12 mg/L air (analytical)
Dose / conc.:
0.36 mg/L air (analytical)
Dose / conc.:
1.08 mg/L air (analytical)
No. of animals per sex per dose:
30
Control animals:
yes, sham-exposed
Maternal examinations:
CAGE SIDE OBSERVATIONS: not specified

DETAILED CLINICAL OBSERVATIONS: Yes
Prior to the exposure period, animals were observed for clinical signs once daily. Preceding and following each exposure, individual does were observed for clinical signs of toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: On gestation day 0, 3, 6 9 12, 15, 18 and 20

FOOD CONSUMPTION: Yes
- The feed consumption by each animal was determined on gestation day 0, 3, 6 9 12, 15, 18 and 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The uteri and ovaries were removed. The uteri were weighed. The number of corpora lutea, implantation sites for each hern of the uterus, dead and live implatations and fetuses were determined.

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:
Living fetuses were weighed, sexed, and examined for externally detectable changes. In addition, attention was paid to the viability of the fetuses, the length of the umbilical cord and the condition of the fetal membranes and amniotic fluid. One-third of the fetuses of each dam were fixed in Bouin's solution and examined for internal soft tissue changes. In order to assess the skeletal system, two-thirds of the living fetuses of each dam were initially fixed in 96% alcohol, then clarified with potassium hydroxide solution and stained with Alizarin red S. The fetuses were stored in 100% glycerol.

- External examinations: Yes: all per dam
- Soft tissue examinations: Yes: one-third per dam
- Skeletal examinations: Yes: two-thirds per dam
Statistics:
Quantitative continuous random variables, eg. body weight data or food consumption data, were examined with the Williams test. Discrete random variables, eg. number of corpora lutea, number of implantations, or percentage values, such as live fetuses as a percentage of total implantations, fetuses with anomalies (or variations, retardations) as a percentage of total fetuses investigated, were examined with a linear rank test of Krauth. The Fisher test was used for the comparison of frequencies, eg. number of litters with anomalies (or variations, retardations) in relation to the number of litters investigated. All tests were performed with type I error of = 0.05 and = 0.01. The linear rank test and the Fisher test were carried out with a Bonferroni correction, but including additional information. If the Bonferroni corrected test did not show any significance for = 0.05, the comparison was calculated with uncorrected = 0.05. The linear rank test and the Fisher test were calculated by counting all possible permutations.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical signs:
No animal died in any group. Indications of a toxic effect on the dams emerged at 120 ppm (decreased consumption of feed during the exposure phase and a smaller difference between the body weight and uterus weight after necropsy). A marked toxicity on the pregnant animals was detected at 360 ppm (lower body weight, body weight gain slowed down, decreased consumption of feed, smaller difference between body weight and uterus weight, and clinical signs of an irritant effect caused by acrylic acid vapours).
Dose descriptor:
NOAEC
Effect level:
0.12 mg/L air (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Abnormalities:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no signs of embryotoxicity, in particular no teratogenic effects.
Dose descriptor:
NOAEC
Effect level:
>= 1.08 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Developmental effects observed:
no

EFFECTS OF EXPOSURES ON MATERIAL BODY WEIGHT AND UTERUS WEIGHT (g)

Concentration (ppm)             

 0

40

120

360

Body weight

Day 0

216 (9.4)a

216 (11.4)

214 (7.7)

215 (9.9)

Day 6

243 (10.1)

243 (16.1)

242 (9.5)

242 (12.7)

Day 15

288 (9.6)

284 (19.2)

280 (14.4)

261 (16.3)**

Day 20

354 (19.9)

349 (33.3)

346 (27.0)

333 (26.1)**

Uterus weight (Day 20)

64 (18.8)

66 (25.5)

68 (23.6)

65 (21.1)

BWE-uterusb

290 (12.0)

283 (17.8)

278 (16.6)**

267 (13.4)**

BWE-BWS-uterusc

74 (11.5)

67 (11.2)*

65 (13.1)**

52 (9.2)**

 aFigures in parentheses indicate standard deviations.

bBWE-uterus = body weight on Day 20 minus uterus weight.

cBWE-BWS-uterus = body weight gain between Day 0 and Day 20 minus uterus weight.

* p<0.05.

**p<0.01.

TERMINAL OBSERVATIONS AND GENERAL FETAL DATA

Concentration (ppm)

0

40

120

360

Number of animals

30

30

29

29

Number of dams

25

27

26

25

Implantations

12.96

12.48

12.46

12.40

Live fetuses

11.92

11.81

11.88

11.68

Dead implantations

1.04

0.67

0.58

0.72

Fetal numbers (male:female)

145:153

140:179

149:160

133:159

Mean weight of live fetuses (g)

3.51

3.64

3.73*

3.77*

Mean length of live fetuses (cm)

3.28

3.30

3.36

3.36

Mean weight of placenta (g)

0.50

0.55

0.52

0.48

 * p<0.05.

FETAL FINDINGS

 

Concentration (ppm)

0

40

120

360

Anomalies/Litters

4 (16.0)m

10 (37.04)

3 (11.54)

3 (13.04)

Anomalies/Fetuses

6 (1.69)n

13 (4.76)

3 (0.81)

3 (1.06)

Variations/Litters

12 (48.00)

17 (62.96)

10 (38.46)

8 (34.78)

Variations/Fetuses

20 (8.04)

36 (11.09)

17 (5.27)

10 (4.36)

Retardations/Litters

24 (96.00)

24 (88.89)

23 (88.46)

21 (91.30)

Retardations/fetuses

114 (35.53)

95 (32.10)

104 (30.63)

89 (32.61)

mFigures in parentheses show percentage of fetuses examined that were affected per litter.

nFigures in parentheses show percentage of fetuses examined that were affected.

Executive summary:

Groups of 30 pregnant Sprague-Dawley rats were exposed (6 h/d, whole-body) to atmospheres containing acrylic acid at 0, 40, 120, and 360 ppm (corresponding to approx. 0, 0.12, 0.36 and 1.08 mg/L) during days 6 to 15 of gestation in a developmental study according to OECD TG 414. After exposure the dams were observed up to day 20 of gestation. The animals’ body weight and food consumption were determined on gestation day 0 and subsequently on every third day up to gestation day 20. After sacrifice dams were subjected to a gross pathological examination. After external examination of each foetus their body weights and lengths were measured and they were further processed for skeletal and visceral examination.

In the dams, irritation of the respiratory tract and the eyes was observed in the highest dose group. A dose-related reduction in food and water intake resulting in a decrease in body weight gain was observed in the 120 and 360 ppm groups. Also in the 40 ppm group a slight but statistically significant effect was seen on body weight gain (between day 0 and 20 minus uterus weight) of the dams (10 % reduction as compared to the control). Since this finding at 40 ppm was the only effect observed at this dose level and with unclear biological relevance, it was concluded that the NOAEC for maternal toxicity was 40 ppm (= approx. 0.12 mg/L).

No effects on reproductive performances were observed. There were no signs of group-related trends or significant differences between groups in terms of pre-implantation losses, live foetuses, or resorptions. There were also no signs of group-related differences in the incidences of abnormalities, variations, or retardations in the foetuses in terms of general appearance, foetal body weights and the conditions of the internal organs or the skeleton. Thus, the NOAEC for developmental toxicity in rats was set at 360 ppm = approx. 1.080 mg/L.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Hazleton Research Products, Inc. (Denver, PA)
- Age at study initiation: 5.5-6 months old
- Weight at study initiation: 2.8-4.0 kg
- Housing: Single in in stainless steel, wire-mesh cages (61 x 61 x 41 cm)
- Diet (ad libitum): AGWAY® PROLAB® Animal Diet (Agway Inc.) Except during exposures
- Water (ad libitum): Tap water (except during exposures)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-21
- Humidity (%): 40-60
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
Females assigned to the study were exposed to acrylic acid vapour or filtered air for 6 hours/day during the period of major organogenesis (gestation day -gd- 6 through 18). Filtered air was bubbled through a glass reservoir containing liquid acrylic acid. For all vapour concentrations, a Dwyer Flowmeter was used to measure airflow prior to passing the air through the acrylic acid. The vapour, was introduced into the exposure chambers through 1 inch glass tubing containing stainless steel wool.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of acrylic acid vapour in each exposure chamber was monitored throughout the 15 days of exposure by sampling with XAD-8 sorbent tubes and subsequent analysis using high performance liquid chromatography (HPLC) analysis. The HPLC system was composed of a Model 981 Lambda Max LC Spectrophotometer, a Programmable Systems 680 Gradient Controller, a 712 WISP, and a Model 501 Solvent Delivery System. A Spectra Physics SP4270 computing Integrator provided a record of the chromatograms, chromatographic analyses, and peak height measurement. The concentration in each exposure chamber atmosphere was determined approximately 3 times during each 6-hour exposure. The control chamber was sampled once daily. The nominal concentration was calculated by dividing the total quantity of acrylic acid delivered to the chamber by the chamber airflow rate.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
Duration of treatment / exposure:
From day 6 to day 18 of gestation
Frequency of treatment:
6 hours/day
Duration of test:
29 days
Dose / conc.:
25 ppm
Remarks:
0.075 mg/L
Dose / conc.:
75 ppm
Remarks:
0.224 mg/L
Dose / conc.:
225 ppm
Remarks:
0.673 mg/L
No. of animals per sex per dose:
16
Control animals:
yes, sham-exposed
Maternal examinations:
CAGE SIDE OBSERVATIONS: not specified

DETAILED CLINICAL OBSERVATIONS: Yes
Prior to the exposure period, animals were observed for clinical signs once daily. Preceding and following each exposure, individual does were observed for clinical signs of toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: Maternal body weights were measured on gd 0, 3, 6, 12, 18, 24, and 29.

FOOD CONSUMPTION: Yes
Food consumption was measured daily throughout the study, beginning on gd 3.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: The gravid uterus, ovaries (including corpora lutea), cervix, vagina, and abdominal and thoracic cavities were examined grossly. The right nasal turbinates were examined. Maternal liver and kidney weights were determined.


Ovaries and uterine content:
Each uterus was removed from the peritoneal cavity, weighed, and dissected longitudinally to expose the contents. All live and dead fetuses and resorption sites (early and late) were recorded. Ovaries were removed from the peritoneal cavity and ovarian corpora lutea of pregnancy were counted. Uteri from females that appeared nongravid were placed in a 10% ammonium sulfide solution for confirmation of pregnancy status.

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:
After removal from the uterus, all live fetuses received a single lethal intraperitoneal injection of sodium pentobarbital. All live and dead fetuses were weighed and examined externally for variations and malformations including cleft palate. All live fetuses in each litter were examined for thoracic and abdominal visceral abnormalities. The sex of each fetus was determined during dissection by examination of the reproductive organs. One-half of the live fetuses in each litter were decapitated. The heads were fixed in Bouin's solution for subsequent examination of craniofacial structures. All fetuses (50% intact, 50% decapitated) in each litter were eviscerated, air-dried, processed for skeletal staining with alizarin red S and examined for skeletal malformations and variations. All fetal skeletal preparations were retained.


- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
The unit of comparison was the pregnant doe or the litter. The data for quantitative continuous variables were intercompared for the 3 exposure groups and the control group by use of Levene's test for equality of variances; analysis of variance (ANOVA), and t-tests. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated similar variances, and the ANOVA was significant, pooled t-tests were used for pairwise comparisons. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by separate variance t-tests for pairwise comparisons.
Nonparametric data were statistically evaluated using the Kruskal-Wallis test, followed by the Mann-Whitney U test when appropriate. Frequency data were compared using Fisher's Exact Test. With the exception of the data analysis for fetal malformations and variations, all statistical analyses were performed using BMDP Statistical Software (Dixon, 1990). For all statistical tests, the probability value of < 0.05 (two-tailed) was used as the critical level of significance.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No animal died in any group. In the 225 ppm group, individual clinical signs observed included: perinasal wetness beginning as early as the first day of exposure and ending by the second day after the last exposure; perinasal encrustation (from days 14 through 22); and nasal congestion observed during and subsequent to exposures (through Day 22). Nasal congestion was observed in a single doe from the 75 ppm group on day 12 only. There were no clinical signs observed in does during or subsequent to exposures to 25 ppm acrylic acid vapour.
Dose descriptor:
NOAEC
Effect level:
0.075 mg/L air (nominal)
Basis for effect level:
other: maternal toxicity
Abnormalities:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There was no evidence of developmental toxicity including teratogenicity at any exposure concentration.
Dose descriptor:
NOAEC
Effect level:
>= 0.673 mg/L air (nominal)
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Developmental effects observed:
no

Maternal food consumption:

Exposure-related decreases in food consumption were observed in the 75 and 225 ppm groups during the first 5 days of the exposure period. Throughout the remainder of the exposure period, daily food consumption was consistently reduced in the 225 ppm group and the decreases occasionally reached statistical significance. Occasional reductions in daily food consumption were also observed during the exposure period subsequent to Day 11 (Days 17 to 19) in the 75 ppm group. Average food consumption/day calculated for the entire exposure period was reduced in the 225, but not 75, ppm group. Statistically significant increases in food consumption were observed subsequent to the exposure period in the 225 ppm group for Days 23 to 24 and 28 to 29 and in the 75 ppm group for Day 23 to 24. Mean food consumption values for Days 24 to 27 suggested a trend toward increased food consumption throughout the post exposure period for the 225 ppm group. Mean values for the 75 ppm group suggest a trend toward increased food consumption through Day 26. The statistically significant reduction (of approximately 16%) in food consumption for Day 8 to 9 in the 25 ppm group was not considered to be biologically significant based on slightly greater food consumption values (including increases of up to 14%) in the low concentration group prior to exposures. Occasional increases or decreases in food consumption values for the 25 ppm group subsequent to Day 9 were not considered to be exposure related due to the lack of a dose-response pattern of effects.

Gestation day

Group mean food consumption (g)

0 ppm

25 ppm

75 ppm

225 ppm

6 – 7

191.42

166.96

147.31**

101.55**

7 – 8

193.42

169.23

149.51**

110.35**

8 – 9

195.13

163.75*

150.69**

121.66**

9 – 10

185.38

166.73

153.59*

132.49**

10 – 11

184.98

200.79

154.79*

134.49**

12 – 13

180.85

123.21*

203.10

136.18**

16 – 17

187.39

186.42

161.23

152.77*

18 – 19

208.56

178.61

167.23*

147.33**

23 - 24

158.19

176.43

204.25**

199.75*

28 - 29

134.35

167.32*

160.41

188.19**

* Significantly different from control group (p<0.05.)

**Significantly different from control group (p<0.01.)

Maternal body weights and weight changes:

Mean gestational body weights were equivalent across groups throughout gestation. There were no statistically significant exposure-related reductions in weight gain. However, mean body weight losses were observed in all acrylic acid-exposed groups for Days 6 to 12. Losses in the 25 ppm group were not considered to be biologically significant, since body weight gains were greater than control values (by approximately 60 g) during the preexposure period and the reductions for Days 6 to 12 were not associated with consistent reductions in food consumption for the first half of the exposure period. Reduced body weight gain values in the 75 and 225 ppm groups for Days 6 to 12 were considered to be an exposure-related effect since the reductions were coincident with consistent reductions in food consumption for the first five days of the exposure period. Likewise, increased body weight gains in the 75 and 225 ppm groups for Days 18 to 29 were associated with increases in food consumption during the postexposure period.

Gestation day

Gestational mean body weight changes (g)

0 ppm

25 ppm

75 ppm

225 ppm

0 - 3

-34.08 (150.76)

-7.47 (152.69)

-6.83 (150.42)

-43.38 (214.46)

3 - 6

203.77 (96.19)

240.45 (76.84)

206.55 (96.92)

163.45 (251.41)

6 - 12

68.43 (65.47)

-18.87 (93.75)

-37.67 (93.47)

-41.06 (201.25)

12 - 18

146.67 (69.62)

160.54 (72.07)

123.51 (63.56)

148.45 (65.59)

18 - 24

112.36 (73.58)

150.13 (109.11)

178.41 (73.84)

176.15 (86.22)

24 - 29

26.65 (89.84)

51.55 (125.51)

64.32 (87.17)

143.49**(96.91)

**Significantly different from control group (p<0.01.)

Numbers in parentheses indicate standard deviation

Maternal Necropsy:

Upon necropsy on Day 29, pertinent findings included ulcerations in the nasal turbinates of 1 female in the 225 ppm group. There were no exposure related changes in mean body weight at sacrifice, gravid uterine weight, corrected body weight, or corrected weight change. There were no significant effects of exposure on relative and absolute kidney or liver weights. (Apparently slight increases in absolute and relative liver weights in the 75 and 225 ppm groups were due to single animals in each group which had abnormally large livers). There were no effects of exposure on the number of ovarian corpora lutea, the number of total, viable, or nonviable (early and late resorptions and dead fetuses) implantations/litter. Although percent preimplantation loss was statistically significantly increased in the mid and high concentration groups, the increases were not concentration dependent. Percent live fetuses and sex ratio were equivalent across groups.

Gestational parametres:

Fetal body weights were unaffected by test substance exposure. There were no increases in the incidences of individual external, visceral or skeletal malformations by category, or of total malformations among all groups. There were no increases in the incidences of individual fetal external, visceral, or skeletal variations, of variations by category, or of total variations among all groups.

Concentration (ppm)

0

25

75

225

Corpora lutea (mean)

8.4

9.1

9.5

8.8

Implantations (mean)

8.6

8.8

8.2

8.5

Viable implants (mean)

8.4

8.6

7.5

7.9

Percent live fetuses (mean)

97.8

98.4

91.3

95.0

Dead implantations (mean)

0.1

0.1

0.0

0.1

Mean weight of live fetuses (g)

43.99

42.44

42.44

42.93

Executive summary:

Groups of 16 pregnant rabbits were exposed (6 h/d, whole-body) to atmospheres containing acrylic acid at 0, 25, 75, and 225 ppm (corresponding to approx. 0.075, 0.224, 0.673 mg/L) during days 6-18 of gestation. All dose groups were observed daily for morbidity and mortality. During the exposure period, animals were observed for clinical signs preceding and subsequent to daily exposures and from outside during actual exposures. Maternal body weights were measured on gestation day 0, 3, 6, 12, 24, and 29. Food consumption was measured daily throughout the study beginning on gestation day 3. After sacrifice on gestation day 29, maternal liver and kidney weights were determined. All foetuses were weighed and examined for external malformations and variations, for thoracic and abdominal visceral abnormalities including internal sex organs, for craniofacial abnormalities and for skeletal malformations and variations.

Dose-related clinical signs (as perinasal/perioral wetness and nasal congestion, as well as reduced body weight gain and food consumption) were observed in the 75 and 225 ppm groups. The overall pregnancy rate was equivalent for all groups (94-100 %). No dose-related effects were observed in the reproduction function of the dams. There were no effects on the number of ovarian corpora lutea, the number of total viable or non-viable (early and late resorptions and dead foetuses) implantations/litter. Percentage live foetuses and sex ratio were equivalent across groups. Foetal body weights were unaffected by test substance exposure. There were no exposure-related increases in the incidences of external, visceral or skeletal malformations or variations.

NOAEC for maternal toxicity was 25 ppm (= approx. 0.075 mg/L).

NOAEC for developmental toxicity: 225 ppm = 0.673 mg/L.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication, comparable to current guidelines
Principles of method if other than guideline:
20-24 pregnant dams/group were exposed by whole-body exposure to concentrations of 50, 100, 200 and 300 ppm acrylic acid vapor (corresponding to approx. 0.15, 0.29, 0.59, and 0.88 mg/L for 6 h/d on gestation days 6-20. Control animals were exposed to filtered room air.*
* Calculation of concentrations (mg/L) based on Derelanko MJ (2000). Toxicologist's Pocket Handbook, CRC Press, conversion table, p. 57
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: IFFA CREDO Breeding Laboratories (Saint-Germain-sur-l' Arbresle, France)
- Age at study initiation: Young, nulliparous females
- Weight at study initiation: 200-220 g
- Housing: Single in clear polycarbonate cages with stainless-steel wire lids and hardwood shaving as bedding.
- Diet: Food pellets (UAR Alimentation Villemoisson, France), ad libitum
- Water: Filtered tap water, ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 5
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
EXPOSURE
Exposures were conducted in 200-L glass/stainless-steel inhalation chambers with dynamic and adjustable laminar air flow (6-20 m3/h). The chamber temperature was set at 23 ± 2°C, and the relative humidity at 50 ± 5 %. The exposure system delivered, with an infusion pump, a constant rate of liquid chemical from the top of a heated glass column filled with glass beads. Compressed air heated by a glass heater was introduced at the bottom of the glass column in a countercurrent fashion to the liquid flow. The vaporized compound was introduced into the main air inlet pipe of the exposure chambers.
Concentrations of acrylic acid were determined 3 times at regular intervals during each 6-h exposure period. Atmosphere samples were collected through quartz fiber filters impregnated with a Na2CO3 and glycerol solution. The filters were then desorbed with deionized water. The resulting samples were analyzed by a Dionex ion chromatograph equipped with AG9-SC and AS9-SC columns.
Due to the low vapor pressure of acrylic acid (3.2 mmHg at 20°C), the presence of liquid particles was evaluated at the highest concentration generated (300 ppm). Airborn particles were measured with an Aerodynamic Particle Sizer (APS 3300 model, TSI, USA), with a minimum detection limit of 0.5 µm. No differences in particle counts were observed between the clean filtered air (control) and the vapor-laden air in the exposure chambers.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical concentrations (mean ± SD):
48.0 ± 5.1 ppm (nominal: 50 ppm)
98.0 ± 9.7 ppm (nominal: 100 ppm)
203.1 ± 19.2 ppm (nominal: 200 ppm)
313.1 ± 34.4 ppm (nominal: 300 ppm)
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2-3
Duration of treatment / exposure:
days 6 to 20 of gestation
Frequency of treatment:
6 hours/day
Duration of test:
until gestation day 21
Dose / conc.:
50 ppm
Remarks:
0.15 mg/L
Dose / conc.:
100 ppm
Remarks:
0.3 mg/L
Dose / conc.:
200 ppm
Remarks:
0.6 mg/L
Dose / conc.:
300 ppm
Remarks:
0.9 mg/L
No. of animals per sex per dose:
20
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale:
Exposure concentrations were based on preliminary studies in which maternal toxicity was observed.
Maternal examinations:
BODY WEIGHT: Yes
- Time schedule for examinations: On gestation day (GD) 0, 6, 13 and 21.


FOOD CONSUMPTION: YES
Food consumption was measured for the intervals GD 6-13 and 13-21.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: The uteri were removed and weighed. The number of implantation sites, resorptions, and dead and live fetuses were recorded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
The number of implantation sites, resorptions, and dead and live fetuses were recorded. Uteri which had no visible implantation sites were stained with ammonium sulfide (10 %) to detect very early resorptions.

Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
Live fetuses were weighed, sexed, and examined for external anomalies including those of the oral cavity. Half of the live fetuses from each litter were preserved in Bouin's solution and examined for internal soft tissue changes. The other half were fixed in ethanol (70 %), eviscerated, and then processed for skeletal staining with alizarin red S for subsequent skeletal examination.

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter
Statistics:
Data were presented as mean ± SD. The number of  implantation sites and live fetuses and the various body weights were analyzed by one-way analysis of variance (ANOVA), followed by Dunnett's test if differences were found. The percentages of non-live  implants and  resorptions and the proportions of fetuses with alterations in each litter were evaluated by using the Kruskal-Wallis test, followed by the Dixon-Massey test where appropriate. Rates of pregnancy, fetal sex ratio, and percentage of litters with malformations or external, visceral, or skeletal variations were analyzed by using Fisher's test. Where applicable, least-squares analysis was carried out. For all statistical tests, the level of significance was set a priori at alpha = 0.05.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
All animals survived the treatment. Maternal body weight gain was significantly reduced during the first half of the exposure period at 200 ppm and throughout the whole exposure period at 300 ppm. Absolute weight gain was significantly reduced at 200 and 300 ppm. Decreased maternal food consumption was observed during the first half of exposure at 50 and 100 ppm (about 8% less than control) and throughout exposure at both higher concentrations. Since exposure to 50 ppm acrylic acid produced only slight maternal toxicity in the form of transient decrease in food consumption without any effect on body weight gain, the NOAEC for maternal toxicity was 50 ppm.
Dose descriptor:
NOAEC
Effect level:
100 ppm
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Abnormalities:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The only finding with respect to developmental toxicity was a concentration-related decrease in fetal body weights that were statistically significantly different from those of the control at 300 ppm (about 9% lower than control). Thus, fetal toxicity occured only in the presence of overt maternal toxicity. No evidence of teratogenic effects was found after exposure up to 300 ppm. Inhaled acrylic acid was not selectively toxic to the embryo or fetus. According to the authours the NOAEC for developmental toxicity was 200 ppm.
Dose descriptor:
NOAEC
Effect level:
200 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
no effects observed
Developmental effects observed:
no

Maternal body weights:

Concentration [ppm/6h/d]

Maternal body weight GD 6 [g]

Absolute weight gain [g]

0

272 ± 16

27 ± 13

50

265 ± 15

29 ± 9

100

269 ± 12

21 ± 12

200

269 ± 14

5 ± 12**

300

268 ± 16

-13 ± 14**

Absolute weight gain: (Day 21 body weight) - (gravid uterus weight) - (Day 6 body weight)

Reproductive parameters:

 

Conc. [ppm/6h/d]

No. of litters

No. of implantation sites/litter

% of non-live implants/litter

% of resorption sites/litter

No. of live fetuses/litter

Average fetal body weight/litter [g]

0

24

14.33 ± 3.60

7.34 ± 20.22

7.34 ± 20.22

14.35 ± 2.69

5.73 ± 0.20

50

20

14.55 ± 3.07

6.72 ± 8.46

6.72 ± 8.46

13.55 ± 3.15

5.72 ± 0.39

100

22

15.23 ± 2.25

8.90 ± 8.68*

8.90 ± 8.68*

13.95 ± 2.75

5.60 ± 0.31

200

21

14.71 ± 3.59

6.78 ± 8.12

6.78 ± 8.12

13.81 ± 3.71

5.58 ± 0.32

300

23

16.00 ± 2.17

9.42 ± 13.27*

8.87 ± 13.13

14.65 ± 2.90

5.22 ± 0.37**

Concentration [ppm/6h/d]

0

50

100

200

300

Mean % of fetuses with:

- any malformations/litter

0.52 ± 1.73

0

0

1.79 ± 5.64

0

- external variations/litter

0

0.33 ± 1.49

0

0

0

- visceral variations/litter

11.82 ± 22.67

23.44 ± 23.57*

10.44 ± 12.95

11.65 ± 17.87

2.61 ± 7.73

- skeletal variations/litter

20 .02 ±17.69

19.06 ± 24.06

14.18 ± 13.07

13.57 ± 15.07

13.25 ± 13.56

- any variations/litter

15.93 ± 16.17

21.79 ± 12.36

12.60 ± 7.0

12.65 ± 11.41

8.02 ± 8.81*

* ,** Significant differences from the control (0 ppm) value, p 0.05, and p 0.01, respectively.

Executive summary:

Saillenfait et al. (1999) exposed groups of 17 - 25 pregnant Sprague-Dawley rats to 0, 50, 100, 200 or 300 ppm acrylic acid for 6 hours/day during gestational days 6 - 20. The concentration in the exposure chamber was analyzed by gas chromatography and was found to be 48.0±5.1, 98.0±9.7, 203.1±19.2 and 313.1±34.4 ppm. Maternal body weight gain was significantly reduced during the first half of gestation at 200 ppm and throughout the whole exposure period at 300 ppm. Absolute weight gain was significantly reduced in groups exposed to 200 ppm or higher. A decrease in maternal food intake was observed during the first half of gestation at 50 and 100 ppm and throughout gestation at higher exposure concentrations. A dose-dependent decrease of fetal body weights was observed, but was significant only in the 300-ppm group. Only sporadic visceral and skeletal malformations were observed. Significant increases of visceral variations occurred in the 50-ppm group, but not in groups exposed to higher acrylic acid concentrations. According to the authors these findings were not related to acrylic acid exposure. The authors did not evaluate possible irritative effects during exposures.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
20-5-2016 to 25-7-2016 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 Jan 2001
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
Aug 1998
GLP compliance:
yes
Specific details on test material used for the study:
- Lot No. F534801GB
- Exp. date: 11 Dec 2016
- Colorless, clear liquid
Species:
rabbit
Strain:
New Zealand White
Remarks:
Hra:(NZW)SPF
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Covance Research Products, Inc., Denver, PA
- Age at study initiation: The animals were approximately 7 months old upon receipt.
- Weight at study initiation: 2922 - 3949 g
- Housing: All rabbits were housed individually in clean, stainless steel cages suspended above ground corncob bedding (Pel-O’Cobs®; The Andersons, Cob Products Division, Maumee, OH). Nesting material was not required because the females were euthanized prior to the date of expected parturition. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly. Kale (1 leaf at each occasion) was provided to each animal daily for environmental enrichment and to aid in maintaining the animal's gastrointestinal health, beginning upon animal receipt and continuing throughout the duration of the study.
- Diet: The basal diet used in this study, PMI Nutrition International, LLC Certified Rabbit LabDiet® 5322, was a certified feed with appropriate analyses performed by the manufacturer. The basal diet was offered in 25-g increments 3 times per day on the day of arrival and in increased amounts over the next few days, until the animals gradually achieved ad libitum status prior to the dose administration period; basal diet was offered ad libitum thereafter.
- Water: Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, was provided ad libitum during the study.
- Acclimation period: 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
other: 1% carboxymethylcellulose (medium viscosity), 0.014% Kolliphor® EL, and 0.0035% hydrochloric acid in deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance formulations were prepared daily as single formulations for each dosage level and maintained on wet ice, protected from light. The test substance formulations were stirred continuously on wet ice throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Concentration in vehicle: 0, 10, 30 and 80 mg/mL (corresponding to dosage levels of 0, 50, 150 and 400 mg/kg/day)
- Amount of vehicle: 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the 10, 30, and 80 mg/mL dosing formulations and from the middle stratum of the control group dosing formulations prepared on on the first, approximate middle, and last days of preparation on which all groups were dosed. Analysis was performed using a validated gas chromatography method using flame ionization detection. The analyzed concentrations was 85% to 115% of the target concentration.
Details on mating procedure:
The time-mated rabbits were received on gestation day 2, 3, or 4; a breeding record was provided by the supplier.
Duration of treatment / exposure:
gestation days 7 through 28
Frequency of treatment:
once daily
Duration of test:
until GD29
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dosage levels were selected based on the results of a previous range-finding study in rabbits.3 In that study, rabbits were dosed from gestation days 7-28 at dosage levels of 50, 125, 250, and 400 mg/kg/day. There were no test substance-related deaths at any dosage level. Lower mean body weight gains (approximately 18%) and food consumption (approximately 19%) combined with decreased defecation were noted in the 400 mg/kg/day group compared to the control group throughout the treatment period (gestation days 7-29). There were no significant clinical observations noted and no treatment-related findings noted at the macroscopic examinations at any dosage level. Based on these results, the following doses were selected for the current study: 400 mg/kg/day as the high-dosage level because it was expected to produce some maternal toxicity (i.e., decreased body weight gain), 150 mg/kg/day as the mid-dosage level, and 50 mg/kg/day as the low-dosage level because it was not expected to cause any toxic effects.
Maternal examinations:
CAGE SIDE OBSERVATIONS
All rabbits were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily from the day of receipt through gestation day 29 (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately 1 hour following dose administration.

BODY WEIGHT
Individual maternal body weights were recorded on gestation days 0 (by supplier under conditions that were not compliant with GLPs, but in accordance with the supplier’s SOPs), 5, and 7-29 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 7-10, 10-13, 13-21, 21-29, and 7-29.

FOOD CONSUMPTION
Individual food consumption was recorded on gestation days 5-29. Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals.

POST-MORTEM EXAMINATIONS
The laparohysterectomies and macroscopic examinations were performed blind to treatment group. All rabbits were euthanized on estation day 29 by an intravenous injection of sodium pentobarbital via the marginal ear vein. The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the postmortem findings were co related with the antemortem observations, and any abnormalities were recorded. Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Gravid uterine weight was collected and net body weight (the gestation day 29 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 0-29 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.
- Number of corpora lutea: The number of corpora lutea on each ovary was recorded.
- Number of implantations: The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in
10% ammonium sulfide solution for detection of early implantation loss.
Fetal examinations:
- Fetal examinations were performed blind to treatment group.
- External examinations: Each viable fetus was examined externally, individually weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Crown-rump measurements, degrees of autolysis and gross examinations, if possible, were recorded for late resorptions, and the tissues were discarded.
- Soft tissue examinations: Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels. The sex of each fetus was determined by internal examination. Fetal kidneys were examined and graded for renal papillae development
- Skeletal examinations: Following fixation in alcohol, each fetus was stained with Alizarin Red S8 and Alcian Blue. Fetuses were then examined for skeletal malformations and developmental variations.
- Head examinations: Heads from approximately one-half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique. The heads from the remaining one-half of the fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol.
Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group.
- Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group.
- Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined), and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group.
Indices:
Postimplantation Loss/Litter = No. Dead Fetuses, Resorptions (Early/Late)/Group / No. Gravid Females/Group

Summation Per Group (%) = Sum of Postimplantation Loss/Litter (%)/ No. Litters/Group

Where: Postimplantation Loss/Litter (%) = No. Dead Fetuses, Resorptions (Early/Late)/Litter/ No. Implantation Sites/Litter x 100

Summation per Group (%) = Sum of Viable Fetuses Affected/Litter (%)/ No. Litters/Group

Where: Viable Fetuses Affected/Litter (%) = No. Viable Fetuses Affected/Litter/ No. Viable Fetuses/Litter x 100
Historical control data:
yes
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related clinical findings were noted at the daily examinations or approximately 1 hour following dose administration at any dosage level. Findings noted in the test substance-treated groups, including decreased defecation and brown material and/or hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All females in the control, 50, 150, and 400 mg/kg/day groups survived to the scheduled necropsy on gestation day 29.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 400 mg/kg/day group, an absence of a mean body weight gain (0 g) was noted on the first day of dose administration (gestation day 7-8) and resulted in a 78.9% lower mean body weight gain in this group compared to the control group during gestation days 7-10 (57 g, 54 g, and 64 g in the control, 50, and 150 mg/kg/day groups, respectively, compared to 12 g in the 400 mg/kg/day group). Mean body weight gains in the 400 mg/kg/day group were similar to the control group for the remainder of the treatment period (gestation days 10-29). As a result of the lower mean body weight gain at the beginning of the treatment period, a lower mean body weight gain was noted at 400 mg/kg/day compared to the control group when the entire treatment period (gestation days 7-29) was evaluated. However, the aforementioned differences were not statistically significant and were not of sufficient magnitude to affect mean body weights at this dosage level, and therefore were considered test substance-related but nonadverse.
Mean maternal body weight gains in the 50 and 150 mg/kg/day groups and mean body weights, net body weights, net body weight gains, and gravid uterine weights in the 50, 150, and 400 mg/kg/day groups were unaffected by test substance administration. Differences from the control group were slight and not statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the 400 mg/kg/day group, lower mean food consumption was noted during gestation days 7 -10 compared to the control group; mean food consumption in this group was similar to the control group for the remainder of the treatment period (gestation days 10-29). As a result of the lower mean food consumption at the beginning of the treatment period, lower mean food consumption was noted in the 400 mg/kg/day group compared to the control group when the entire treatment period (gestation days 7-29) was evaluated. However, the aforementioned differences at 400 mg/kg/day were not statistically significant and were not of sufficient magnitude to affect mean body weights at this dosage level, and therefore were considered test substance-related but nonadverse.
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 50 and 150 mg/kg/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
At the scheduled necropsy on gestation day 29, no test substance-related internal findings were observed at dosage levels of 50, 150, and 400 mg/kg/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Dead fetuses:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related external malformations were noted for fetuses in this study. In the 150 mg/kg/day group, one fetus was noted with omphalocele (a portion of the liver protruded through an opening in the umbilicus, remnants of a membranous sac). The aforementioned malformation noted at 150 mg/kg/day occurred in a single fetus, did not occur in a dose-related manner, and the mean litter proportion was not statistically significantly different from the concurrent control group and was within the test lab historical control data range (version 2016.01); therefore, it was not considered test substance-related. No external developmental variations were observed in fetuses in this study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- No test substance-related skeletal malformations were noted for fetuses at any dosage level. Vertebral anomaly with or without an associated rib anomaly (extra or fused ribs; extra and/or malpositioned arches; extra, malpositioned, absent, small, fused, and/or misshapen centra) was noted for 2 and 1 fetuses in the 50 and 150 mg/kg/day groups, respectively. The aforementioned malformation at 50 and 150 mg/kg/day occurred infrequently, did not occur in a dose-related manner, and the mean litter proportions were not statistically significantly different from the concurrent control group and were within the Charles River Ashland historical control data range; therefore, it was not considered test substance-related.
- No test substance-related skeletal developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- No test substance-related visceral malformations were observed for fetuses at any dosage level. Lobular agenesis of the lungs (right accessory lobe absent) was noted for and 3 fetuses in the 50 and 150 mg/kg/day groups, respectively. Because this finding occurred infrequently, did not occur in a dose-related manner, and the mean litter proportions were not statistically significantly different from the concurrent control group and were within the Charles River Ashland historical control data range, this finding was not considered test substance-related. In the control group, one fetus was noted with an absent left kidney and ureter and one fetus was noted with an absent right thyroid gland.
- No test substance-related visceral developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.
- A distended stomach was noted for one fetus in the 50 mg/kg/day group. This finding was not classified as either a malformation or developmental variation, was not considered to be test substance-related because it occurred infrequently and in a manner that was not dose-related.
Details on embryotoxic / teratogenic effects:
The numbers of fetuses (litters) available for morphological evaluation were 219(25), 214(24), 199(25), and 214(24) in the control, 50, 150, and 400 mg/kg/day groups, respectively. Malformations were observed in 2(1), 4(4), 5(4), and 0(0) fetuses (litters) in these same respective dosage groups and were considered spontaneous in origin.
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
Nonadverse lower mean body weight gains and corresponding lower mean food consumption were noted in the 400 mg/kg/day group. No evidence of developmental toxicity was noted at 50, 150, and 400 mg/kg/day. Based on these results, a dosage level of 400 mg/kg/day, the highest dosage level tested, was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and prenatal developmental toxicity when n-butyl acrylate was administered orally by gavage to time-mated New Zealand White rabbits.
Executive summary:

The potential of n-butyl acrylate to induce developmental toxicity in rabbits after maternal exposure from implantation to 1 day prior to expected parturition was evaluated in an OECD TG # 414 study by oral route.

n-Butyl acrylate, in the vehicle (1% carboxymethylcellulose [medium viscosity], 0.014% Kolliphor®EL, and 0.0035% hydrochloric acid in deionized water) was administered orally by gavage to 3 groups of 25 time-mated female New Zealand White [Hra:(NZW)SPF] rabbits once daily from gestation days 7 through 28. Dosage levels were 50, 150, and 400 mg/kg/day administered at a dose volume of 5 mL/kg. A concurrent control group of 25 time-mated females received the vehicle on a comparable regimen. The females were approximately 7 months of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 29, a laparohysterectomy was performed on each female. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

All females in the control, 50, 150, and 400 mg/kg/day groups survived to the scheduled necropsy. No test substance-related clinical observations were noted at the daily examinations or approximately 1 hour following dose administration at any dosage level.

A test substance-related absence of a mean body weight gain (0 g) was noted in the 400 mg/kg/day group on the first day of dose administration (gestation day 7-8) and resulted in a 78.9% lower mean body weight gain in this group compared to the control group during gestation days 7-10 and a lower mean body weight gain when the entire treatment period (gestation days 7-29) was evaluated. However, the aforementioned differences were not statistically significant and were not of sufficient magnitude to affect mean body weights at this dosage level, and therefore were considered test substance-related but nonadverse. In addition, lower mean food consumption was noted in the 400 mg/kg/day group during gestation days 7-10 and resulted in lower mean food consumption in this group compared to the control group when the entire treatment period (gestation days 7-29) was evaluated; however, these differences were not statistically significant and were not of sufficient magnitude to affect mean body weights at this dosage level, and therefore were considered test substance-related but nonadverse. Mean maternal body weights, body weight gains, and food consumption in the 50 and 150 mg/kg/day groups and mean body weights, net body weights, net body weight gains, and gravid uterine weights in the 50, 150, and 400 mg/kg/day groups were unaffected by test substance administration. There were no substance-related macroscopic findings noted at the scheduled necropsy on gestation day 29 in the 50, 150, and 400 mg/kg/day groups. Intrauterine growth and survival in the 50, 150, and 400 mg/kg/day groups were unaffected by test substance administration. In addition, no test substance-related external, visceral, and skeletal malformations or developmental variations were noted at any dosage level.

Nonadverse lower mean body weight gains and corresponding lower mean food consumption were noted in the 400 mg/kg/day group. No evidence of developmental toxicity was noted at 50, 150, and 400 mg/kg/day. Based on these results, a dosage level of 400 mg/kg/day, the highest dosage level tested, was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and prenatal developmental toxicity when n-butyl acrylate was administered orally by gavage to time-mated New Zealand White rabbits.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication, comparable to current guidelines
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
Groups of 20-29 bred female rats (17-25 pregnant) were exposed to the compound 6h/day on days 6 through 20 of gestation. Control animals were exposed concurrently to filtered room air. The test concentrations of n-butyl acrylate were 100, 200, and 300 ppm (corresponding to approx. 0.52, 1.05, and 1.57 mg/L)*.
* Calculation of concentrations (mg/L) based on Derelanko MJ (2000). Toxicologist's Pocket Handbook, CRC Press, conversion table, p. 57
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: IFFA CREDO Breeding Laboratories (Saint-Germain-sur-l' Arbresle, France)
- Age at study initiation: Young, nulliparous females
- Weight at study initiation: 200-220 g
- Housing: Single in clear polycarbonate cages with stainless-steel wire lids and hardwood shaving as bedding.
- Diet: Food pellets (UAR Alimentation Villemoisson, France), ad libitum
- Water: Filtered tap water, ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 5
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
EXPOSURE
Exposures were conducted in 200-L glass/stainless-steel inhalation chambers with dynamic and adjustable laminar air flow (6-20 m3/h). The chamber temperature was set at 23 ± 2°C, and the relative humidity at 50 ± 5 %. The air-flow rate passed through the fritted disk of a heated bubbler containing the test chemical. Concentrations of acrylate ester were monitored continuously with a gas-chromatograph equipped with a flame ionization detector and an automatic gas-sampling valve. In addition, exposure levels were determined once during each 6-h exposure period by collecting atmosphere samples through glass tubes packed with activated charcoal. The charcoal samples were then desorbed with carbon disulfide. The resulting samples were analyzed by gas chromatography using appropriate internal standards.


GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Glass/stainless-steel inhalation chambers
- Source and rate of air: Test atmospheres were generated through an additional air-flow  rate passed through the fritted disk of a heated bubbler containing ethylhexyl acrylate. The vaporized compound was introduced into the main air inlet pipe of the exposure chamber.
- Air flow rate: 6-20 m3/h


TEST ATMOSPHERE
- Brief description of analytical method used: GC/FID
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical concentrations (mean ± SD):
103.3 ± 6.7 ppm (nominal: 100 ppm)
202.8 ± 9.7 ppm (nominal: 200 ppm)
302.5 ± 10.1 ppm (nominal: 300 ppm)
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2-3
- Length of cohabitation: Overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
days 6 to 20 of gestation
Frequency of treatment:
6 hours/day
Duration of test:
until gestation day 21
Dose / conc.:
100 ppm
Remarks:
0.52 mg/L
Dose / conc.:
200 ppm
Remarks:
1.05 mg/L
Dose / conc.:
300 ppm
Remarks:
1.57 mg/L
No. of animals per sex per dose:
20
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale:
Exposure concentrations were based on preliminary studies in which marked decreases in maternal weight gain were observed at 200 and 300 ppm of butyl acrylate. Results from previous prenatal inhalation toxicity studies on butyl acrylate were also considered (Merkle and Klimisch, 1983). The high concentrations of butyl acrylate for the definitive study (200 and 300 ppm, respectively) were chosen to maximize the opportunity of identifying embryolethal or teratogenic potential.
Maternal examinations:
BODY WEIGHT: Yes
- Time schedule for examinations: On gestation day (GD) 0, 6, 13 and 21.


FOOD CONSUMPTION: YES
Food consumption was measured for the intervals GD 6-13 and 13-21.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: The uteri were removed and weighed. The number of implantation sites, resorptions, and dead and live fetuses were recorded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
The number of implantation sites, resorptions, and dead and live fetuses were recorded. Uteri which had no visible implantation sites were stained with ammonium sulfide (10 %) to detect very early resorptions.

Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
Live fetuses were weighed, sexed, and examined for external anomalies including those of the oral cavity. Half of the live fetuses from each litter were preserved in Bouin's solution and examined for internal soft tissue changes. The other half were fixed in ethanol (70 %), eviscerated, and then processed for skeletal staining with alizarin red S for subsequent skeletal examination.

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter
Statistics:
Data were presented as mean ± SD. The number of  implantation sites and live fetuses and the various body weights were analyzed by one-way analysis of variance (ANOVA), followed by Dunnett's test if differences were found. The percentages of non-live  implants and  resorptions and the proportions of fetuses with alterations in each litter were evaluated by using the Kruskal-Wallis test, followed by the Dixon-Massey test where appropriate. Rates of pregnancy, fetal sex ratio, and percentage of litters with malformations or external, visceral, or skeletal variations were analyzed by using Fisher's test. Where applicable, least-squares analysis was carried out. For all statistical tests, the level of significance was set a priori at alpha = 0.05.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
All female rats survived the test. The average maternal body weight gain (gd 6 - 21) was 141, 128, 84 and 18 g for the 0; 100, 200 and 300 ppm dose groups, respectively. These maternal weight gains were markedly lower in the 200 and 300 ppm dose groups as compared to the controls (p < 0.01). Absolute weight gain of dams, expressed as the day 21 body weight minus the gravid uterus weight and minus the day 6 body weight, was significantly reduced in the 100*, 200** and 300** ppm groups, respectively (* for p<0.05; ** for p<0.01), with the absolute weight gains reported to bei 32, 18, -16 and -60 g for the 0, 100, 200 and 300 ppm groups, respectively.
A NOAEC was not observed for maternal toxicity. The LOAEC for maternal toxicity was 100 ppm (0.52 mg/L/day).
Dose descriptor:
LOAEC
Effect level:
100 ppm
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Abnormalities:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
No treatment-related effects were reported in terms of numbers of implanation sites, live fetuses, non-live implants or resorptions. Fetal body weight was significantly reduced at 200 ppm (for both sexes combined and males) and at 300 ppm (both sexes combined, males and females). These decreases amounted to 7-8 % (p<0.05) and 26-28 % (p<0.01) of control values for the 200 and 300 ppm groups, respectively. A few sporadic malformations were seen in the 300 ppm and the control group. There was no evidence of treatment-related effects on the incidence of external and visceral variations. The incidence of individual skeletal variations (mainly incomplete ossification of sternebrae and of vertebral centra) was similiar in the control and treated groups.
The NOAEC for fetotoxicity was 100 ppm (0.52 mg/L/day).
The NOAEC for teratogenicity was 300 ppm (1.57 mg/L/day; highest dose tested).
Dose descriptor:
NOAEC
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
no effects observed
Developmental effects observed:
no

Maternal body weights:

Concentration [ppm/6h/d]

Maternal body weight GD 6 [g]

Absolute weight gain [g]

0

294 ± 23

32 ± 15

100

289 ± 23

18 ± 14*

200

299 ± 24

-16 ± 20**

300

292 ± 23

-60 ± 26**

Absolute weight gain: (Day 21 body weight) - (gravid uterus weight) - (Day 6 body weight)

Reproductive parameters:

Conc. [ppm/6h/d]

No. of litters

No. of implantation sites/litter

% of non-live implants/litter

% of resorption sites/litter

No. of live fetuses/litter

Average fetal body weight/litter [g]

0

25

15.68 ± 3.17

10.9 ± 15.49

10.64±15.62

14.12 ± 4.01

5.74 ± 0.43

100

24

15.58 ± 3.05

6.82 ± 10.19

6.82 ± 10.19

14.71 ± 3.57

5.71 ± 0.35

200

24

15.08 ± 4.23

4.72 ± 5.96

4.72 ± 5.96

14.46 ± 4.20

5.33 ± 0.40*

300

25

15.40 ± 5.24

6.48 ± 15.94

6.48 ± 15.94

14.68 ± 5.38

4.25 ± 0.94**

Concentration [ppm/6h/d]

0

100

200

300

Mean % of fetuses with:

- any malformations/litter

2.00 ± 7.33

0

0

0.62 ± 2.65

- external variations/litter

1.33 ± 6.67

0

0

0.22 ± 1.11

- visceral variations/litter

8.81 ± 14.64

0

0

4.17 ± 20.41

- skeletal variations/litter

13.70 ± 15.48

17.01 ± 14.53

18.71 ± 24.21

24.65 ± 20.69

- any variations/litter

12.60 ± 10.80

13.27 ± 15.07

10.10 ± 10.32

15.90 ± 19.98

* ,** Significant differences from the control (0 ppm) value, p 0.05, and p 0.01, respectively.

Executive summary:

In a study, groups of 20-29 pregnant female Sprague Dawley rats were exposed to n-butyl acrylate concentrations of 100, 200 and 300 ppm (0.53; 1.06; 1.6 mg/l) for 6 hours per day on days 6 through 20 of gestation.

A NOAEL for maternal toxicity could not be determined since the absolute weight gain was significantly reduced in all dose groups in a concentration-related manner. No treatment-related effects were reported in terms of numbers of implantation sites, live fetuses, non-live implants or resorptions. Fetal body weight was significantly reduced at 200 and at 300 ppm. A few sporadic malformations were seen in the 300 ppm and the control group. There was no evidence of treatment-related effects on the incidence of external and visceral variations. The incidence of individual skeletal variations (mainly incomplete ossification of sternebrae and of vertebral centra) was similiar in the control and treated groups.

The NOAEL for maternal toxicity could not be identified; the LOAEL was 100 ppm. The NOAEL for developmental toxicity was 100 ppm and the NOAEL for teratogenicity was 300 ppm.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
14 Feb 1979 - 13 Mar 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
other: Guidelines for reproduction studies for safety evaluation of drugs for human use, FDA, Jan. 1966 and Guidance on reproduction studies from the Association of the British Pharmaceutical Industry, 1975.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Iffa Credo, Lyon, France
- Body weight at study initiation:
The mean body weight ± SD in dose groups 0, 25, 135 and 250 ppm were 209±12, 214±14, 214±9 and 219±16, respectively.
- Age at study initiation: 9-11 week
- Diet (e.g. ad libitum): Herilan Mrh-Zucht, H. Eggermann KG, Rinteln.
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±5
- Photoperiod (hrs dark / hrs light):12/12


Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
By means of a continuous infusion apparatus (UNITA I, B. Braun, Melsungen, Federal Republic Germany) constant amounts of the liquid product were supplied to a heated (about 80°C) evaporator. The n-butyl acrylate vapors were diluted with dust-free, conditioned fresh air and passed through 200 L inhalation chambers (all-glass construction with steel frame) under dynamic airflow conditions at a flow rate of 20 changes of air per hour (4000 L/h; 200 L chamber). A mean temperature of 24.5°C and a mean relative humidity of 53% were measured during exposure.


TEST ATMOSPHERE
- Brief description of analytical method used: The n-butyl acrylate test atmosphere concentrations were monitored analytically by means of a total
hydrocarbon analyzer (R 5 of RATFISCH, Munich). The total hydrocarbon analyzer was calibrated using an infrared analyzer Miran I (WILKS) calibrated with standards of known concentrations of n-butyl acrylate.
- Samples taken from breathing zone: yes


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical concentrations (Mean ± SD) of the dose groups 25, 135 and 250 ppm were 25 ± 1, 137 ± 4 and 251 ± 3 ppm, respectively.
Details on mating procedure:
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
days 6-15 of gestation
Frequency of treatment:
6 hours per day
Duration of test:
21 days
Remarks:
Doses / Concentrations:
0, 25, 135 and 250 ppm (corresponding to 0, 0.13, 0.71 and 1.31 mg/L) Calculation of concentrations (mg/L) based on Derelanko MJ (2000). Toxicologist's Pocket Handbook, CRC Press, conversion table, p. 57.
Basis:
nominal conc.
No. of animals per sex per dose:
30
Control animals:
yes, sham-exposed
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: day on which sperm had been detected (day 0) and on the 6th, 16th and 20th days post coitum.



POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on the 20th day post coitum.



Ovaries and uterine content:
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
Fetal examinations:
The weights and the length of fetuses were determined. After fixation in Bouin's solution, 1/3 of the fetuses were examined for organ changes according to the method of Wilson and Warkany (1965), and after staining of the skeleton (Dawson, 1926) 2/3 of the fetuses were investigated for skeletal changes.
Wilson, J.G. and Warkany, J. (1965). Teratology: Principles and Techniques. The University of Chicago Press, Chicago and London.
Dawson, A.B. (1926). Stain Technol. 1:123.
Statistics:
A trend analysis based on the generalization of the t-test according to Williams (1971, 1972) was carried out for the variables of maternal body weight and body weight gain, fetal weight and length, and placental weight in each case. The U-test (Krauth, 1971; Stucky and Vollmar, 1976) was carried out for the parameters of implantations per pregnant animal, live and dead embryos as percent per pregnant animal, and anomalies, variations and retardations as percent of live fetuses per litter.
Williams, D.A. (1971). Biometrics 27:103-117.
Williams, D.A. (1972). Biometrics 28:519-531.
Krauth, J. (1971). Ann. Math. Statist. 42:1949-1956.
Stucky, W. and Vollmar, J. (1976). J. Statist. Comput. Simul. 5:73-81.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
25 ppm were tolerated without any impairment of body weight. The body weight gain was significantly reduced after inhalation of 135 and 250 ppm during the period of treatment. In the period after the end of treatment (gd 16 - 20) the steepness of the body weight curve obtained after 135 and 250 ppm was similar to that of the control group. During the exposure 135 ppm led to distinct discharge from the eyes and noses and to ruffled fur. After inhalation of 250 ppm these symptoms were even more pronounced. No mortality occurred.
It is noteworthy to mention that no histopathological examination on the respiratory tract of the dams was performed, as it was not required by the guideline. However, with regards to the observed respiratory irritation in the subchronic inhalation study at comparable concentrations (see 7.5.2 BASFAGXXVI/352), the occurrence of clear effects on the respiratory tract as evidence for maternal toxicity can be considered as very likely, especially as irritation was already noted clinically.
Dose descriptor:
NOAEC
Effect level:
ca. 0.13 mg/L air (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
early or late resorptions
Abnormalities:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The necropsy of the animals did not reveal any gross-pathological changes of the internal organs which could be attributed to the test substance. The number of corpora lutea and the number of implantations did not show any differences between the individual groups. After inhalation of 135 and 250 ppm the percentage of resorptions (i.e. post-implantation loss) per pregnant animal was dose-dependent reduced. No adverse effect on the weight and length of the fetuses was observed. No treatment related morphological alterations, especially no malformations were observed in the fetuses at any concentration.
Dose descriptor:
NOAEC
Effect level:
ca. 0.13 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
Abnormalities:
no effects observed
Developmental effects observed:
yes
Lowest effective dose / conc.:
0.71 mg/L air (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Maternal body weight development (mean values ± standard deviation):

 

Concentration [ppm]

Maternal body weight GD 0 [g]

Maternal body weight GD 20 [g]

Maternal body weight gain GD 0-20 [g]

0

209.38 ± 11.83

354.01 ± 36.66

144.64 ± 33.08

25

213.96 ± 13.94

359.62 ± 36.81

145.66 ± 29.87

135

213.68 ± 9.30

335.54 ± 43.00

121.87 ± 37.62*

250

218.64 ± 16.19

318.11 ± 42.79**

99.47± 33.68**

*  p < 0.05

** p < 0.01

Reproductive parameters:

 

Conc.  (ppm)   

no. pregnant/  total animals 

live fetuses/ animal 

resorptions (%)

weight of fetuses (g)

0

22/30

11.5 ± 5.34     

11.6

3.85 ± 0.41

25

23/30

10.6 ± 4.94     

13.8

4.08 ± 0.39

135

18/30

8.8 ± 5.14        

23.6*

4.09 ± 0.23

250

19/30

8.4 ± 5.68        

31.6*

4.08 ± 0.47

*: p<0.05

Conc.  (ppm)

% fetuses per litter with anomalies

% litters with fetuses showing anomalies

% fetuses per litter with variations/ retardations

% litters with fetuses showing variations/ retardations

0

2.7

23.8

19.7

81.0

25

0.9

9.1

11.2

59.1

135

1.9

18.8

10.2

43.8

250

0

0

8.2

43.8

 

0 ppm

25 ppm

135 ppm

250 ppm

Skeletal findings

Anomalies:

- Cleft vertebral centra

7/170

2/162

4/105

0/107

Variations/retardations:

- incomplete ossification of skull bone

0/170

0/162

1/105

0/107

- aplasia of sternebrae

16/170

11/162

3/105

3/107

-incomplete ossification of sternebrae

30/170

18/162

14/105

9/107

- asymmetric sternebrae

4/170

1/162

0/105

0/107

-accessory rib, bilateral

2/170

3/162

0/105

0/107

-accessory rib, bilateral and rudimentary

1/170

0/162

0/105

0/107

-accessory rib, unilateral and rudimentary

1/170

0/162

0/105

0/107

-general incomplete ossification of bones

1/170

2/162

1/105

1/107

Organ findings

Variations/retardations:

-dilatation of pelvis, unilateral

2/82

0/81

1/54

0/53

Units given as number found/number examined

 

Executive summary:

Sprague Dawley rats were exposed to n-butyl acrylate concentrations of 25, 135 and 250 ppm for 6 hours per day on days 6 to 15 of gestations (BASF AG, 1979).

 

Inhalation of 135 and 250 ppm of the test substance caused a significant reduction in maternal body weight gain, as well as irritation to the nose and eyes. At the end of the exposure period, the weight gain was comparable to the controls.  The two highest exposure concentrations caused embryo lethality, as evidenced by a dose-dependent increase in post-implantation loss. The 25-ppm dose did not lead to any signs of maternal toxicity or embryo lethality. No signs of organ changes or skeletal abnormalities were observed in the fetuses at any concentration.

 

The NOAEL for maternal and developmental toxicity was 25 ppm, and the NOAEL for teratogenicity was 250 ppm.

 

In 1999 study, groups of 20-29 pregnant female Sprague Dawley rats were exposed to n-butyl acrylate concentrations of 100, 200 and 300 ppm for 6 hours per day on days 6 through 20 of gestation (Saillenfait, 1999).

 

A NOAEL for maternal toxicity could not be determined since the absolute weight gain was significantly reduced in all dose groups in a concentration-related manner. No treatment-related effects were reported in terms of numbers of implantation sites, live fetuses, non-live implants or resorptions. Fetal body weight was significantly reduced at 200 and at 300 ppm. A few sporadic malformations were seen in the 300 ppm and the control group. There was no evidence of treatment-related effects on the incidence of external and visceral variations. The incidence of individual skeletal variations (mainly incomplete ossification of sternebrae and of vertebral centra) was similiar in the control and treated groups.

 

The NOAEL for maternal toxicity was < 100 ppm, the NOAEL for developmental toxicity was 100 ppm and the NOAEL for teratogenicity was 300 ppm.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Adopted according to OECD SIDS (publicly available peer reviewed source). Only summary report available. Low reliability due to excessive dose levels causing maternal lethality and by far exceeding the recommended maximum dose level to be used according to OECD guidelines..
Principles of method if other than guideline:
Groups of bred female mice were administered with the test compound through oral gavage route on days 5 through 20 of gestation. Control animals received concurrently the vehicle. The doses of n-butyl acrylate were 100, 1000, 1500, 2000, 2500, 3000, 4000 mg/kg bw/day.
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
CD-1
Route of administration:
oral: gavage
Vehicle:
cotton seed oil
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/3
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
day 6-15 of gestation
Frequency of treatment:
daily
Duration of test:
no data
Remarks:
Doses / Concentrations:
0; 100, 1000; 1500; 2000; 2500; 3000; 4000 mg/kg bw in cottonseed oil
Basis:
actual ingested
No. of animals per sex per dose:
25-30
Control animals:
yes, concurrent vehicle
Maternal examinations:
BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, 6, 11, 15 and 18 of gestation.


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
Fetal examinations:
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No animal survived at the high dose. At 3000 and 2500 mg/kg bw 2/30, at 2000 mg/kg bw 1/29, at 1500 mg/kg bw 1/27 and at 1000 mg/kg bw 1/ 30 died respectively. At 1500 mg/kg bw and above the mean maternal body weight gain was reduced.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Basis for effect level:
body weight and weight gain
mortality
Abnormalities:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Fetal body weights were reduced at doses of 1500 mg/kg bw and above. In the 2500, 3000 mg/kg bw and higher dose groups, the percentage of resorptions was significantly increased. A significant dose response trend was observed for "average percent malformed fetuses". Dose groups of 1000 mg/kg bw and above produced significant effects on this parameter. In the control group, at 100 mg/kg, 1000 mg/kg, 1500 mg/kg and 2000 mg/kg variations and malformations occurred sporadically and on different sides in a non-dose-dependent manner (i.e. cleft palate, fused ribs, fused sternebrae, fused arches, extra arches, branched ribs), with a slight dose-dependent increase when taking the sum of all events per dose group together. In the 2500 mg/kg and 3000 mg/kg dose groups, dose levels which caused severe maternal toxicity in form of body weight reductions up to mortalities, the number of fetuses with external and skeletal malformations and variations (cleft palate, exencephaly, open eyes, fused archs, fused ribs) was significantly increased.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
visceral malformations
Abnormalities:
effects observed, treatment-related
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 500 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

The summaries on maternal toxicity, developmental efffects including morphological alterations and up to teratogenicity were taken from the SIDS report since the complete study report was not available.

Executive summary:

In a gavage study, pregnant CD-1 mice were administered n-butyl acrylate dissolved in cottonseed oil. Concentrations of 0, 100, 1000, 1500, 2000, 2500, 3000 and 4000 mg n-butyl acrylate/kg bw were administered from gestation day 6 to 15. No animals survived in the high dose group. At 3000 and 2500 mg/kg, 2 of 30 animals died; at 2000 mg/kg 1 of 29 died; at 1500 mg/kg, 1 of 27 died; and at 100 mg/kg, 1 of 30 died. At the 1500 mg/kg dose and higher, average maternal body weight gain was significantly reduced.

 

Fetal body weights were significantly reduced at doses of 1500 mg/kg and above. At 2500 and 3000 mg/kg, the percentage of resorptions was significantly increased.

 

At 100 mg/kg, 1000 mg/kg, 1500 mg/kg and 2000 mg/kg, and in the control group, variations and malformations occurred sporadically on different sides (i.e. single cases of cleft palate, fused ribs, fused sternebrae, fused arches, extra arches, branched ribs) in a non-dose-dependent manner, with a slight dose-dependent increase when taking the sum of all events per dose group together. In the 2500 mg/kg and 3000 mg/kg groups, the number of fetuses with external and skeletal malformations and variations (cleft palate, exencephaly, open eyes, fused arch’s, fused ribs) was significant increased (Rohm and Haas Co., 1982).

 

Taking maternal mortality and reduced weight gain into account; the NOAEL for maternal toxicity was 100 mg/kg. The NOAEL for developmental toxicity was 1000 mg/kg and the NOAEL for teratogenicity was 2000 mg/kg.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
191 mg/kg bw/day
Study duration:
subacute
Experimental exposure time per week (hours/week):
168
Species:
rabbit
Quality of whole database:
Reliability 1; OECD 414 in rabbits under GLP
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
750 mg/m³
Study duration:
subacute
Experimental exposure time per week (hours/week):
42
Species:
rat
Quality of whole database:
Reliability 2, well performed and documented study
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

An OECD TG #414 in rabbits as requested in the Compliance Check Decision number: CCH-D-2114340871-51-01/F, was performed - draft report available.

2-Ethylhexyl acrylate - Prenatal Developmental Toxicity Study in New Zealand White Rabbits Administration in the diet (ARTF 2023)

In a prenatal developmental toxicity study, 2-Ethylhexyl acrylate was administered to pregnant New Zealand White rabbits daily as a homogeneous addition to the food from GD 6-29 at target dose levels of

50, 150 and 300 mg/kg bw/d (nominal).

Under the conditions of this prenatal developmental toxicity study, the oral administration of 2 -Ethylhexyl acrylate as homogeneous inclusion in the diet to pregnant New Zealand White rabbits from implantation to the expected day of

parturition (GD 6-29) caused evidence of beginning maternal toxicity (food refusal because of stomach irritation) at the highest dose. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is

effectively 119 mg/kg bw/d.

Since there was no evidence for treatment-related adverse effects of the test substance on fetal morphology the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is effectively 191 mg/kg bw/d.

It is notable, that a number of females across all dose groups experienced abortions or complete litter losses after very long time periods of either food refusal or distinctly reduced food consumption. These effects occurred

independent of the applied test substance concentrations in the diet and had a profound influence on average test substance intake of their respective treatment groups. If these outliers were discounted from the calculation

of test substance intake (= effective dose), the corresponding group means were about 42 mg/kg bw/d, 131 mg/kg bw/d and 225 mg/kg bw/d for the low-, mid- and high dose group, respectively.

The test substance is not teratogenic in rabbits at the tested dose levels.

 

2 -Ethylhexyl acrylate - OECD 414 in rats: Relative Developmental Toxicities of  Acrylates in Rats following Inhalation Exposure (Saillenfait 1999)

Groups of 23 to 25 dams were exposed (6 hours/day, whole-body) to atmospheres containing 2-ethylhexyl acrylate (99.7% purity) at 0, 50, 75, and 100 ppm (approximately 0.375, 0.563, and 0.750 mg/l) during day 6 to day 20

of gestation.

In summary, no embryotoxic, teratogenic or fetotoxic properties of 2-EHA had been revealed from this study for concentrations of up to and including 100 ppm. Based on slightly reduced food intake and lower maternal weight

gain at the higher exposure level a NOAEC/maternal toxicity of 75 ppm (approximately 0.563 mg/l) is derived from this study. No embryo-/fetotoxic effects were revealed even at the highest tested concentration at which some

signs of maternal toxicity had been observed. Therefore, a NOAEC/developmental toxicity of 100 ppm (approximately 0.750 mg/l) is derived from this study.

 

2-ethylhexanol

The toxicity to reproduction (effects on fertility and developmental toxicity) of 2-ethylhexanol was evaluated by the eMS Poland as a part of the substance evaluation process under the REACH Regulation (EC) No 1907/2006.

One of the reasons for the selection of 2-EH was health hazard concern. It was noted in the screening that developmental effects were observed in the pre-natal developmental toxicity studies. Therefore, a full evaluation

of the available information was required in order to assess whether the observed developmental effects are the result of maternal toxicity. This part of the document reviews the available study reports in order to assess

reproductive toxicity of 2-EH, and assess whether a proposal of harmonised C&L is needed for this endpoint.

Excerpt from the CoRAP evaluation report (2015) related to the developmental toxicity:

•The results of animal studies provide evidence of an adverse effect of 2-EH on development at very high doses [> 1000 mg/kg bw/d] causing strong toxic effects in dams, therefore they can be considered as a secondary

non-specific consequence of other toxic effects. Evaluation of the available information shows that no maternal toxicity or slight maternal toxicity was observed with in animal studies and no developmental toxicity warranting

classification is observed.

It was concluded that this concern could be removed because outcome of a full evaluation of the available information shows that doses of 2-EH are not toxic or are only slightly toxic to maternal animals and no developmental

toxicity warranting classification is observed.

There are several studies to evaluate developmental toxicity of 2-EH: two studies in mice (Tyl et al., 1991; Hardin et al. 1987) and four studies in rats (Hellwig and Jäckh, 1997; Ritter et al. 1987, Tyl et al. 1992, Nelson et al. 1989).

Oral route

2-EH was examined in a mouse feeding study for its potential for developmental toxicity equivalent to OECD TG 414 and under GLP conditions (Tyl et al., 1991). Timed pregnant female CD-1 Swiss mice (28 animals/group,

body weight range 32.5 to 31.6 g) received 2-EH in the diet at nominal concentrations of 0, 0.009, 0.03, and 0.09% during gestation days 0-17. The animals were housed singly and observations for clinical signs were made

daily. Body weights were recorded on gestational day 0, 3, 6, 9, 12, 15, 17. Food consumption and test compound intake were calculated individually. Test substance purity and concentration in the diets was verified using gas

chromatography. Test substance purity was >99%. Concentration in the diets was within 99-108% of the nominal concentration.

Food intake and hence dose levels were higher than expected. Average intakes were 0, 17, 59, and 191 mg/kg bw/day, respectively. No dams died, delivered early or were removed from study. Pregnancy rates were high

(93-96%) and equivalent across all groups. One litter at 0% was fully resorbed; all other pregnant animals had live litters at scheduled necropsy. The numbers of live litters evaluated were 27 at 0.009 and 0.03% and

26 at 0 and 0.09% 2-EH.

There was no treatment-related maternal toxicity observed in this study. Maternal body weights, weight gains (absolute or corrected for gravid uterine weight), gravid uterine weight and liver weight (absolute or relative to body

weight) were unaffected. Food consumption (g/kg/day and g/day) was significantly increased for GD 0-3 at 0.09% and unaffected for all other time points evaluated. The calculated consumption of 2-EH, based on gestational

food consumption was 0 (0 mmol/kg), 17 (0.13 mmol/kg), 59 (0.46 mmol/kg) and 191 mg/kg/day (1.49 mmol/kg), for the 0, 0.009, 0.03 and 0.090% groups, respectively.

There were no effects of exposure to dietary 2-EH on any of the gestational parameters. The number of corpora lutea, uterine implantation sites (live, dead, resorbed), pre- and post-implantation loss, sex ratio and live fetal

body weight per litter (all fetuses or separately by sex) were all equivalent across all groups. There were also no treatment-related changes in the incidence of individual, external, visceral, skeletal or total malformations or

variations. 

In conclusion, there were no maternal or developmental toxic effects of 2-EH dietary exposure throughout gestation at any concentration tested. The NOAEL for maternal toxicity and for developmental toxicity and teratogenicity

was therefore 191 mg/kg bw/day, the highest dose level tested.

The developmental toxicity of 2-EH was furthermore investigated in a comparative and thus limited toxicity study according to OECD TG 414 study (Hellwig and Jäckh, 1997). The study addressed all the endpoints required in

the OECD TG 414 and was conducted under GLP, but used low rat numbers (due to the fact that the study purpose was a comparative screening for several alcohols; only 10 instead of 20 pregnant females). This invalidates

the study to some extent, but it provides weight of evidence for the developmental toxicity endpoint. Animals were treated on GD6 to 15 via gavage at dose levels of 130, 650, and 1300 mg/kg bw/d. Controls were either treated

with water only or with vehicle (i.e. 0.005% Cremophor EL).

Maternal toxicity was most severe at the high dose level including mortality (6/10 animals). In the mid and low dose no relevant maternal toxicity was noted. Therefore the NOAEL is 650 mg/kg/d for this endpoint under the

conditions of this study.

Signs of embryo-/fetotoxicity were dose-dependently noted in dams showing signs of maternal toxicity at 1300 mg/kg/d. The observed effects (decreased mean fetal bw, increase of skeletal variations and retardation) in the

mid-dose group were invalidated when compared to historical controls or current expert opinion in agreement with Regulation (EC) No 1272/2008 was applied:

- the decreased body weights were within the range of historical controls

- original statistical evaluation of the study was assessed to be invalid (reevaluation was performed)

- within this statistical reevaluation (Wilcoxon test; based on litter data) the statistical significance of skeletal variations was not confirmed

- skeletal retardations were still significant

- however skeletal retardations of control goups also exceeded the historical control values

- and in agreement with current expert opinion and Regulation (EC) No 1272/2008 skeletal retardations can be considered as transient non-adverse effects.

Therefore the NOAEL is 650 mg/kg/d for this endpoint under the conditions of this study.

Teratogenicity was noted in fetuses from the high dose dams only. Therefore the NOAEL is 650 mg/kg/d under the conditions of this study for teratogenicity.

Dermal route

The developmental toxicity of 2-EH following dermal absorption was examined in a OECD TG 414 rat study that was conducted under GLP (Tyl et al., 1992). 2-EH was applied to the skin of 25 females at 0, 252, 840, and 2520

mg/kg bw/day under an occlusive dressing during gestational days 6 -15 for 6 hours per day. The dose levels were selected based on the results of a preliminary study.

The maternal toxicity was mild. There were no deaths or severe clinical signs of toxicity. A reduced body weight gain in high-dose rats was noted and local skin irritation in rats at the intermediate and the high dose level.

2-EH had no adverse effect on the maternal gestational parameters, or maternal organ weights, or on the fetal weight, sex ratio, viability, or the incidence of malformations and variations.

Therefore, the NOAEL for maternal systemic toxicity was 840 mg/kg bw/day, based on the effects on body weight gain; the NOAEL for skin irritation was 252 mg/kg bw/day. The NOAEL for developmental toxicity and teratogen

icity was 2520 mg/kg bw/day.

Inhalation route

No maternal toxicity or developmental toxicity was noted in a rat inhalation study that was conducted similar to OECD TG 414; the study is valid though less animals (n = 15) than suggested by the test guideline (Nelson et al., 1989).

The rats were exposed during days 0-19 of gestation to 2-EH at 850 mg/m³.

There were only minor signs of maternal toxicity, i.e. reduction of feed consumption (p<0.05) and body weight gain during gestation. The reproduction parameters were unchanged. There was no indication of developmental

toxicity or teratogenicity, as the incidences of resorptions, the number of fetuses per litter, the sex ration, fetal weight, were all comparable to the control group. Moreover, there were no external, skeletal or visceral

malformations observed in any group.

The authors concluded that due to the low vapor pressure the systemic dose was too low to produce maternal or developmental toxicity. This does not only apply for 2-EH, but also for other alcohols with chain lengths of 5 or

more carbons. Smaller alcohols with chain lengths of 1 to 4 carbons are much more volatile, and produce maternal toxicity, and also developmental toxicity in the presence of maternal toxicity.

The inhalation NOAEC for maternal toxicity and developmental toxicity and teratogenicity was therefore 850 mg/m³.

eMS’s conclusion: The results of animals studies provide evidence of an adverse effect of 2-EH on development at very high doses causing strong toxic effects in dams, therefore they can be considered as a secondary

non-specific consequence of other toxic effects. Evaluation of the available information shows that no maternal toxicity or slight maternal toxicity was observed with in animal studies and no developmental toxicity warranting

classification is observed.

 

Acrylic acid

No prenatal developmental toxicity was observed (rats and rabbits, inhalation), even at concentration levels that produced some signs of maternal toxicity. No specific teratogenic potential could be revealed for dose levels up

to and including 360 ppm (rats) (= approx. 1.08 mg/L) and 225 ppm (rabbits) (= approx. 0.673 mg/L), respectively. According to the present database acrylic acid does not show any potential to cause toxicity to development.

 

Inhalation route

Inhalation exposure of pregnant rats and rabbits to atmospheres containing acrylic acid at concentrations up to 360 ppm (rats) and 225 ppm (rabbits) produced no evidence of developmental toxicity in either species.

 

Rat

Groups of 30 pregnant Sprague-Dawley rats were exposed (6 h/d, whole-body) to atmospheres containing acrylic acid at 0, 40, 120, and 360 ppm (corresponding to approx. 0, 0.12, 0.36 and 1.08 mg/L) during days 6 to 15 of

gestation in a developmental study according to OECD TG 414. After exposure the dams were observed up to day 20 of gestation (BASF, 1983). The animals’ body weight and food consumption were determined on gestation

day 0 and subsequently on every third day up to gestation day 20. After sacrifice dams were subjected to a gross pathological examination. After external examination of each fetus their body weights and lengths were

measured and they were further processed for skeletal and visceral examination.

In the dams, irritation of the respiratory tract and the eyes was observed in the highest dose group. A dose-related reduction in food and water intake resulting in a decrease in body weight gain was observed in the 120 and

360 ppm groups. Also in the 40 ppm group a slight but statistically significant effect was seen on body weight gain (between day 0 and 20 minus uterus weight) of the dams (10 % reduction as compared to the control). Since

this finding at 40 ppm was the only effect observed at this dose level and with unclear biological relevance, it was concluded that the NOAEC for maternal toxicity was 40 ppm (= approx. 0.12 mg/L).

No effects on reproductive performances were observed. There were no signs of group-related trends or significant differences between groups in terms of pre-implantation losses, live foetuses, or resorptions. There were

also no signs of group-related differences in the incidences of abnormalities, variations, or retardations in the foetuses in terms of general appearance, foetal body weights and the conditions of the internal organs or the

skeleton. Thus, the NOAEC for developmental toxicity in rats was set at 360 ppm = approx. 1.080 mg/L.

Saillenfait et al. (1999) exposed groups of 17 - 25 pregnant Sprague-Dawley rats to 0, 50, 100, 200 or 300 ppm acrylic acid for 6 hours/day during gestational days 6 - 20. The concentration in the exposure chamber was analyzed by gas chromatography and was found to be 48.0±5.1, 98.0±9.7, 203.1±19.2 and 313.1±34.4 ppm. Maternal body weight gain was significantly reduced during the first half of gestation at 200 ppm and throughout the whole exposure period at 300 ppm. Absolute weight gain was significantly reduced in groups exposed to 200 ppm or higher. A decrease in maternal food intake was observed during the first half of gestation at 50 and 100 ppm and throughout gestation at higher exposure concentrations. A dose-dependent decrease of fetal body weights was observed, but was significant only in the 300-ppm group. Only sporadic visceral and skeletal malformations were observed. Significant increases of visceral variations occurred in the 50-ppm group, but not in groups exposed to higher acrylic acid concentrations. According to the authors these findings were not related to acrylic acid exposure. The authors did not evaluate possible irritative effects during exposures.

 

Rabbit

Groups of 16 pregnant rabbits were exposed (6 h/d, whole-body) to atmospheres containing acrylic acid at 0, 25, 75, and 225 ppm (corresponding to approx. 0.075, 0.224, 0.673 mg/L) during days 6-18 of gestation

(Neeper-Bradley et al., 1993, 1997). All dose groups were observed daily for morbidity and mortality. During the exposure period, animals were observed for clinical signs preceding and subsequent to daily exposures and

from outside during actual exposures. Maternal body weights were measured on gestation day 0, 3, 6, 12, 24, and 29. Food consumption was measured daily throughout the study beginning on gestation day 3. After sacrifice

on gestation day 29, maternal liver and kidney weights were determined. All fetuses were weighed and examined for external malformations and variations, for thoracic and abdominal visceral abnormalities including internal

sex organs, for craniofacial abnormalities and for skeletal malformations and variations.

Dose-related clinical signs (as perinasal/perioral wetness and nasal congestion, as well as reduced body weight gain and food consumption) were observed in the 75 and 225 ppm groups. The overall pregnancy rate was

equivalent for all groups (94-100 %). No dose-related effects were observed in the reproduction function of the dams. There were no effects on the number of ovarian corpora lutea, the number of total viable or non-viable

(early and late resorptions and dead foetuses) implantations/litter. Percentage live foetuses and sex ratio were equivalent across groups. Foetal body weights were unaffected by test substance exposure. There were no

exposure-related increases in the incidences of external, visceral or skeletal malformations or variations.

NOAEC for maternal toxicity was 25 ppm (= approx. 0.075 mg/L).

NOAEC for developmental toxicity: 225 ppm = 0.673 mg/L.

Thus, inhalation exposure of pregnant rats and rabbits to atmospheres containing acrylic acid at concentrations up to 360 ppm (rats) and 225 ppm (rabbits) produced no evidence of developmental toxicity in either species.

 

n-Butyl acrylate

After oral gavage at very high doses (2500 mg/kg) to mice, n-butyl acrylate caused malformations, only in the presence of maternal toxicity. At concentrations where maternal toxicity was not observed, n-butyl acrylate did not

cause developmental toxicity or teratogenicity. In an oral developmental toxicity study in rabbits, a NOAEL of 400 mg/kg bw was derived for both maternal and developmental toxicity based on the absence of adverse effects.

After inhalation, n-butyl acrylate caused fetotoxic effects in rats only at high maternal toxic doses but no teratogenicity occurred.

 

Oral route

Mouse

In a gavage study, pregnant CD-1 mice were administered n-butyl acrylate dissolved in cottonseed oil (Rohm and Haas Co., 1982). Concentrations of 0, 100, 1000, 1500, 2000, 2500, 3000 and 4000 mg n-butyl acrylate/kg bw

were administered from gestation day 6 to 15. No animals survived in the high dose group. At 3000 and 2500 mg/kg, 2 of 30 animals died; at 2000 mg/kg 1 of 29 died; at 1500 mg/kg, 1 of 27 died; and at 1000 mg/kg, 1 of 30

died. At the 1500 mg/kg dose and higher, average maternal body weight gain was significantly reduced. Fetal body weights were significantly reduced at doses of 1500 mg/kg and above. At 2500 and 3000 mg/kg, the

percentage of resorptions was significantly increased. At 100 mg/kg, 1000 mg/kg, 1500 mg/kg and 2000 mg/kg, and in the control group, variations and malformations occurred sporadically on different sides (i.e. single

cases of cleft palate, fused ribs, fused sternebrae, fused arches, extra arches, branched ribs) in a non-dose-dependent manner, with a slight dose-dependent increase when taking the sum of all events per dose group

together. In the 2500 mg/kg and 3000 mg/kg groups, the number of fetuses with external and skeletal malformations and variations (cleft palate, exencephaly, open eyes, fused arch’s, fused ribs) was significant increased.

Taking maternal mortality and reduced weight gain into account; the NOAEL for maternal toxicity was 100 mg/kg. The NOAEL for developmental toxicity was 1000 mg/kg and the NOAEL for teratogenicity was 2000 mg/kg.

 

Rabbit

The potential of n-butyl acrylate to induce developmental toxicity in rabbits after maternal exposure from implantation to 1 day prior to expected parturition was evaluated in an OECD TG # 414 study by oral route. n-Butyl acrylate,

in the vehicle (1% carboxymethylcellulose [medium viscosity], 0.014% Kolliphor® EL, and 0.0035% hydrochloric acid in deionized water) was administered orally by gavage to 3 groups of 25 time-mated female New Zealand

White [Hra:(NZW)SPF] rabbits once daily from gestation days 7 through 28. Dosage levels were 50, 150, and 400 mg/kg/day administered at a dose volume of 5 mL/kg. A concurrent control group of 25 time-mated females

received the vehicle on a comparable regimen. The females were approximately 7 months of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical

observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 29, a laparohysterectomy was performed on each female. The uteri, placentae, and ovaries were examined,

and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. 

The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

All females in the control, 50, 150, and 400 mg/kg/day groups survived to the scheduled necropsy. No test substance-related clinical observations were noted at the daily examinations or approximately 1 hour following

dose administration at any dosage level. A test substance-related absence of a mean body weight gain (0 g) was noted in the 400 mg/kg/day group on the first day of dose administration (gestation day 7-8) and resulted

in a 78.9% lower mean body weight gain in this group compared to the control group during gestation days 7-10 and a lower mean body weight gain when the entire treatment period (gestation days 7-29) was evaluated. 

However, the aforementioned differences were not statistically significant and were not of sufficient magnitude to affect mean body weights at this dosage level, and therefore were considered test substance-related

but nonadverse. In addition, lower mean food consumption was noted in the 400 mg/kg/day group during gestation days 7-10 and resulted in lower mean food consumption in this group compared to the control group

when the entire treatment period (gestation days 7-29) was evaluated; however, these differences were not statistically significant and were not of sufficient magnitude to affect mean body weights at this dosage level,

and therefore were considered test substance-related but nonadverse. Mean maternal body weights, body weight gains, and food consumption in the 50 and 150 mg/kg/day groups and mean body weights, net body

weights, net body weight gains, and gravid uterine weights in the 50, 150, and 400 mg/kg/day groups were unaffected by test substance administration. There were no substance-related macroscopic findings noted

at the scheduled necropsy on gestation day 29 in the 50, 150, and 400 mg/kg/day groups. Intrauterine growth and survival in the 50, 150, and 400 mg/kg/day groups were unaffected by test substance administration. 

In addition, no test substance-related external, visceral, and skeletal malformations or developmental variations were noted at any dosage level.

Nonadverse lower mean body weight gains and corresponding lower mean food consumption were noted in the 400 mg/kg/day group. No evidence of developmental toxicity was noted at 50, 150, and 400 mg/kg/day. 

Based on these results, a dosage level of 400 mg/kg/day, the highest dosage level tested, was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and prenatal developmental toxicity

when n-butyl acrylate was administered orally by gavage to time-mated New Zealand White rabbits.

 

Inhalation route

Sprague Dawley rats were exposed to n-butyl acrylate concentrations of 25, 135 and 250 ppm (0.13; 0.72; 1.33 mg/l) for 6 hours per day on days 6 to 15 of gestations (BASF AG, 1979).

Inhalation of 135 and 250 ppm of the test substance caused a significant reduction in maternal body weight gain, as well as irritation to the nose and eyes. At the end of the exposure period, the weight gain was comparable

to the controls. The two highest exposure concentrations caused embryo lethality, as evidenced by a dose-dependent increase in post-implantation loss. The 25-ppm dose did not lead to any signs of maternal toxicity or

embryo lethality. No signs of organ changes or skeletal abnormalities were observed in the fetuses at any concentration.

The NOAEL for maternal and developmental toxicity was 25 ppm, and the NOAEL for teratogenicity was 250 ppm.

In a study, groups of 20-29 pregnant female Sprague Dawley rats were exposed to n-butyl acrylate concentrations of 100, 200 and 300 ppm (0.53; 1.06; 1.6 mg/l) for 6 hours per day on days 6 through 20 of gestation

(Saillenfait, 1999).

A NOAEL for maternal toxicity could not be determined since the absolute weight gain was significantly reduced in all dose groups in a concentration-related manner. No treatment-related effects were reported in terms

of numbers of implantation sites, live fetuses, non-live implants or resorptions. Fetal body weight was significantly reduced at 200 and at 300 ppm. A few sporadic malformations were seen in the 300 ppm and the control

group. There was no evidence of treatment-related effects on the incidence of external and visceral variations. The incidence of individual skeletal variations (mainly incomplete ossification of sternebrae and of

vertebral centra) was similiar in the control and treated groups.

The NOAEL for maternal toxicity could not be identified; the LOAEL was 100 ppm. The NOAEL for developmental toxicity was 100 ppm and the NOAEL for teratogenicity was 300 ppm.

Justification for classification or non-classification

GHS classification: GHS UN Rev. 9, 2021 identical to REGULATION (EC) No 1272/2008: no classification required

Additional information