2-Propenoic acid, reaction products with pentaerythritol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January to September 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to standard guidelines in compliance with GLP.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

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, USA
- Age at study initiation: ca. 63 d
- Housing: stainless steel wire mesh cages suspended above cage board
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (e.g. ad libitum): yes
- Acclimation period: 17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.8 - 22.7°C
- Humidity (%): 38.2 - 45.1%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 January 2010 To: 19 May 2010

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Concentration in vehicle: 0, 5, 15 or 40 mg/ml
- Amount of vehicle (if gavage): 5ml/kg bw
- Lot/batch no. (if required): YF0793, YR1134, and YJ0917

Details on mating procedure:

The animals were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females. A breeding record containing the male and female identification numbers and the start date of cohabitation was maintained. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in plastic maternity cages.

For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Fifteen-day room temperature resuspension homogeneity and stability of the test substance formulated in the vehicle at concentrations of 10 and 200 mg/mL were established in a previous study (Stump, Draft, WIL-738003). Therefore, resuspension homogeneity and stability were not assessed for the 15 and 40 mg/mL formulations prepared for the current study.

Prior to the initiation of dose administration, quadruplicate samples for homogeneity and concentration determination were collected from the top, middle, and bottom strata of the 5 mg/mL non-dosing formulation. However, because the analytical results of the initial samples as well as the back-up samples did not meet the WIL Research Laboratories, LLC’s SOP requirements, a new 5 mg/mL non-dosing formulation was prepared. Quadruplicate samples for homogeneity and concentration determination were collected from the top, middle, and bottom strata of the new 5 mg/mL non-dosing formulation. In addition, quadruplicate samples for resuspension homogeneity and stability determinations were collected from aliquots prepared from this same non-dosing formulation following room temperature storage for 5 and 13 days; the aliquots were stirred for at least 60 minutes prior to sampling. Quadruplicate samples for homogeneity and concentration analyses were collected from the top, middle, and bottom of the test substance formulations prepared for the first week of dose administration; samples were also collected from the middle stratum of the vehicle control formulation. Additionally, quadruplicate samples for concentration analysis were collected from the middle stratum of each dosing formulation and vehicle control formulation prepared for the remainder of the study. One set of duplicate samples from each collection was subjected to the appropriate analyses. The remaining set of duplicate samples was stored frozen (approximately -70°C ± 5°C) as back-up. All analyses were conducted by the Analytical Chemistry Department at WIL Research Laboratories, LLC using a validated high performance liquid chromatography method with ultraviolet absorbance detection.

Duration of treatment / exposure:

The males were dosed once daily during study Days 0-27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses. The females were dosed once daily during study Days 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 4) for a total of 40-47 doses. Females that failed to deliver were dosed through the day prior to euthanasia (post-mating Day 25) for a total of 41 doses.

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 25, 75 or 200 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

1é males and 12 females

Control animals:

yes

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. A detailed physical examination was conducted weekly on each animal beginning approximately 1 week prior to the initiation of dose administration, including on the day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual male body weights were recorded beginning approximately 1 week prior to the initiation of dose administration, on the first day of dose administration and weekly thereafter until the day prior to scheduled euthanasia. Individual female body weights were recorded beginning approximately 1 week prior to the initiation of dose administration, on the first day of dose administration and weekly thereafter until the day evidence of copulation was observed.

FOOD CONSUMPTION:
- Individual food consumption was recorded on the corresponding weekly body weight days until pairing. Food intake was not recorded during the mating period. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4. Following mating, food consumption for the female with no evidence of mating was measured on a weekly basis until parturition. When food consumption could not be determined for an animal during a given interval (due to an unscheduled death, as females enter gestation/lactation, weighing error, food spillage, obvious erroneous value, etc.), group mean values were calculated for that interval using the available data.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy (Study Day 28 for males and Lactation Day 5 for females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 6 animals/sex/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy (Study Day 28 for males and Lactation Day 5 for females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 6 animals/sex/group

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- FOB assessments were recorded for 6 animals/sex/group prior to dose administration and fasting for clinical pathology sampling on study day 27 (males) and lactation day 4 (females).
- Locomotor activity counts were recorded for 6 animals/sex/group prior to dose administration on study day 27 (males) and on lactation day 4 (females); the same animals evaluated for FOB were selected for locomotor activity assessment.

GROSS PATHOLOGY

HISTOPATHOLOGY

Litter observations:

LITTER VIABILITY AND DEATHS
Each litter was examined daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained. Intact offspring dying were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984). The carcass of each pup was then discarded.

LITTER CLINICAL OBSERVATIONS
Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1 and 4. Any abnormalities in nursing behavior were recorded.

LITTER BODYWEIGHTS
Pups were individually weighed on PND 1 and 4. Mean pup weights were presented by sex for each litter and by dose group. When body weights could not be determined for a pup during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods a given pup was not weighed were left blank or designated as “NA” on the individual report tables.

LITTER SEX DETERMINATION
Pups were individually sexed on PND 0 (if possible), 1, and 4.

Clinical signs:

effects observed, treatment-related

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:

effects observed, treatment-related

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

CLINICAL OBSERVATIONS AND SURVIVAL

Test substance-related mortality and/or moribundity were noted in the 75 mg/kg bw/day group (2 males) and the 200 mg/kg bw/day group (2 males and 3 females). Clinical findings noted for the majority of the animals that did not survive to the scheduled necropsy included gasping, rales, limbs cool to touch, cool body, labored respiration, mucoid feces, salivation, and red, yellow, and clear material primarily around the mouth; single animals were also noted with wiping mouth in the bedding, splayed limbs, and tonic convulsions at the post-dosing observations. Although the cause of death of death/moribundity of these animals was not determined upon microscopic examination, all animals were noted with lesions in the stomach and lymphoid depletion or lymphoid necrosis in the thymus and generally in the spleen. All other animals survived to the scheduled necropsies.

For animals that survived to the scheduled necropsy, salivation-related findings (salivation prior to or at time of dosing and clear material around the mouth and nose) were noted for the majority of the animals in the 75 and 200 mg/kg bw/day groups primarily at the time of dose administration or approximately 1 h following dose administration; these findings were also occasionally noted for the 25 mg/kg bw/day group animals and were likely signs of taste aversion related to the irritating properties of the compound that resulted in stomach findings and were not considered adverse. Furthermore, the majority of the females in the 25, 75, and 200 mg/kg bw/day groups were noted to be wiping their mouth in the bedding following dose administration due to the irritative nature of the test substance formulations. Incidences of yellow and red material primarily around the mouth were also noted sporadically across all dosage levels during the treatment period, and incidences of rales were noted for a few animals primarily in the 75 and 200 mg/kg bw/day groups at the time of dose administration and/or approximately 1 h following dose administration. These findings were likely related to aspiration of the test substance formulations.
Other clinical findings noted at the daily examinations, at the time of dose administration, or approximately 1 h following dose administration, including hair loss on various body surfaces, occurred infrequently and/or at similar frequencies in the control group, and were not attributed to test substance administration.

BODY WEIGHTS

Test substance-related lower mean body weight gains were noted in the 200 mg/kg bw/day group throughout the treatment period (study Day 0-27); the difference from the control group achieved significance (p<0.01) during study Day 7-13. As a result, mean body weight gains in this group were significantly (p<0.01) lower when the overall pre-mating period (study Day 0-13) and treatment period (study Day 0-27) were evaluated and mean body weights were 7.1% to 8.8% lower during study Day 13-27 compared to the control group; differences in mean body weights were significant (p<0.05) on study Day 21 and 27.
In the 75 mg/kg bw/day group males, slightly (not statistically significant) lower mean body weight gains were noted during study Day 0-21. During study Day 21-27, mean body weight gain in this group was markedly (not statistically significant) lower primarily due to 2 animals (male) noted with body weight losses of 29 g to 34 g during this interval. However, the differences in mean body weight gains were not of a sufficient magnitude to result in statistically significant differences when the overall pre-mating (study Day 0-13) and treatment (study Day 0-27) periods were evaluated and mean body weights in this group were similar to the control group values throughout the treatment period.

Mean male body weights and body weight gains in the 25 mg/kg bw/day group were similar to the control group throughout the treatment period. Differences from the control group were slight and not statistically significant.

FEMALES
Mean female body weights and body weight gains in the 25, 75, and 200 mg/kg bw/day groups were similar to the control group during the pre-mating period (study Day 0-13). Differences from the control group were slight and not statistically significant.

GESTATION
Mean body weights and body weight gains in the 25, 75, and 200 mg/kg bw/day groups were unaffected by test substance administration during gestation. A significantly (p<0.05) lower mean body weight gain was noted in the 200 mg/kg bw/day group during gestation Day 7-11 compared to the control group. However, this difference was transient and therefore, not attributed to test substance administration. In the 75 mg/kg bw/day group, mean body weight gain was significantly (p<0.05) lower when the overall gestation period (gestation Day 0-20) was evaluated. In the absence of a dose-related trend, this difference was not attributed to test substance administration. All other differences in mean body weight gains during gestation were slight and not statistically significant when the test substance-treated groups were compared to the control group.

LACTATION
Mean body weights and body weight gains in the 25, 75, and 200 mg/kg bw/day groups were similar to the control group during lactation Day 1-4. Differences from the control group were slight and not statistically significant.

FOOD CONSUMPTION

MALES
Mean male food consumption, evaluated as g/animal/day and g/kg/day, in the 200 mg/kg bw/day group, was slightly reduced during the pre-mating period (study Day 0-13). Although differences from the control group did not achieve statistical significance, the lower mean food consumption in this group corresponded to reductions in mean body weight gains during this period.
Mean male food consumption in the 25 and 75 mg/kg bw/day groups was similar to the control group during the pre-mating period (study Day 0-13). Differences from the control group were slight and not statistically significant.

FEMALES
Mean female food consumption in the 25, 75, and 200 mg/kg bw/day groups was similar to the control group throughout the pre-mating period (study Day 0-13).

GESTATION
Mean food consumption in the 25, 75, and 200 mg/kg bw/day groups was unaffected by test substance administration during gestation. Significantly (p<0.05) lower mean food consumption was noted in the 200 mg/kg bw/day group during gestation Day 14-17 (g/animal/day and g/kg/day) and when the overall gestation period (gestation Day 0-20; g/kg/day only) was evaluated.

LACTATION
Mean food consumption in the 25, 75, and 200 mg/kg bw/day groups was unaffected by test substance administration during lactation Day 1-4. Differences from the control group were slight and not statistically significant.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB)

HOME CAGE OBSERVATIONS
Home cage parameters were unaffected by test substance administration at all dosage levels. There were no statistically significant differences for the test substance-treated groups when compared to the control group on study Day 27 (males) or lactation Day 4 (females).

HANDLING OBSERVATIONS
Handling parameters were unaffected by test substance administration at all dosage levels. There were no statistically significant differences for the test substance-treated groups when compared to the control group on study Day 27 (males) or lactation Day 4 (females).

OPEN FIELD OBSERVATIONS
Open field parameters were unaffected by test substance administration at all dosage levels. A significant (p<0.01) increase in mean defecation counts was noted in the 25 mg/kg bw/day group females (1.7 counts) compared to the control group (0.0 counts) on lactation Day 4. However, in the absence of a dose-related response, this difference was not attributed to test substance administration. There were no other statistically significant differences for the test substance-treated groups when compared to the control group on study Day 27 (males) or lactation Day 4 (females).

SENSORY OBSERVATIONS
Sensory parameters were unaffected by test substance administration at all dosage levels. There were no statistically significant differences for the test substance-treated groups when compared to the control group during on study Day 27 (males) or lactation Day 4 (females).

NEUROMUSCULAR OBSERVATIONS
Neuromuscular parameters were unaffected by test substance administration at all dosage levels. There were no statistically significant differences for the test substance-treated groups when compared to the control group on study Day 27 (males) or lactation Day 4 (females).

PHYSIOLOGICAL OBSERVATIONS
A lower (10.3%) mean body weight was noted for the 200 mg/kg bw/day group males compared to the control group at the physiological observations on study Day 27. Although this difference did not achieve statistical significance, the lower mean body weight corresponded to the lower weekly mean body weights recorded for this group during the treatment period. No other differences in physiological parameters were attributed to test substance administration at any dosage level. There were no statistically significant differences for the test substance-treated groups when compared to the control group on study Day 27 (males) or lactation Day 4 (females).

LOCOMOTOR ACTIVITY
Locomotor activity patterns (total activity as well as ambulatory activity counts) were unaffected by test substance administration at all dosage levels when evaluated on study Day 27 (males) and lactation Day 4 (females). Values obtained from the 6 subintervals evaluated (0-10, 11-20, 21-30, 31-40, 41-50 and 51-60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the WIL historical control data (Version 1.3). Differences from the control group were slight, not statistically significant when analyzed by a repeated measures analysis, within the WIL historical control data ranges, and/or did not occur in a dose-related manner. No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups.

CLINICAL PATHOLOGY
Higher mean absolute neutrophil counts were noted in the 75 and 200 mg/kg bw/day group males (approximately 149% and 185%, respectively) and females (approximately 46% and 118%, respectively). The difference for the 200 mg/kg bw/day group females achieved significance (p<0.01). These higher neutrophil counts were consistent with the test substance-related ulcerations and associated inflammation that was present in the non-glandular stomach.
There were no other test substance-related effects on hematology or coagulation parameters. Significant (p<0.05 or p<0.01) differences from the control group included lower mean red blood cell counts and hemoglobin values in the 25 and 200 mg/kg bw/day group males and a lower hematocrit in the 25 mg/kg bw/day group males. These group mean differences were not considered to be test substance-related because the values did not show a dose-related response. Mean absolute reticulocyte count for the 200 mg/kg bw/day group males was higher (not statistically significant) than the 0 mg/kg bw/day group males but the value was within the range of historical control data (version 3.1). Significant (p<0.05 or p<0.01) findings that involved percentage reticulocyte or leukocyte differential counts were not itemized above and were not considered toxicologically important because absolute cell counts are more relevant for interpretative purposes.

SERUM CHEMISTRY
There were no test substance-related effects on serum chemistry parameters. There were significantly (p<0.05 or p<0.01) lower mean alkaline phosphatase values in the 75 (101±7.5 U/L) and 200 mg/kg bw/day (92±6.6 U/L) group F0 males but values were within the range of historical controls (version 3.1). In addition, a significantly (p<0.01) higher mean cholesterol value was noted for the 200 mg/kg bw/day group males (65±3.1 mg/dL). These group mean differences were not considered to be test substance-related.

REPRODUCTIVE PERFORMANCE
No test substance-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance-treated/exposed groups. Males that did not sire a litter numbered 1, 1, 0, and 0 in the control, 25, 75, and 200 mg/kg bw/day groups, respectively. Females that had evidence of mating but did not deliver numbered 1, 1, 0, and 0 in the same respective groups.
The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value. None of these differences were statistically significant.

GESTATION LENGTH AND PARTURITION
Mean gestation lengths in the 25, 75, and 200 mg/kg bw/day groups were unaffected by test substance administration. A significantly (p<0.01) longer gestation length was noted for the 75 mg/kg bw/day group (22.0 d) compared to the concurrent control group (21.4 d). However, the magnitude of difference was small, there was no dose-related response, and the value for the 75 mg/kg bw/day group was within the range of the historical control data (21.5-22.3 d). Therefore, this difference was not attributed to test substance administration.

ANATOMIC PATHOLOGY

MACROSCOPIC EXAMINATIONS
In the 200 mg/kg bw/day group, one male was found dead on study 15 and three female were found dead on study Day 3, study Day 16, and gestation Day 13, respectively. In addition, two males in the 75 mg/kg bw/day group were euthanized in extremis on study Day 10 and 13, respectively, and one male in the 200 mg/kg bw/day group was euthanized in extremis on study Day 26. The majority of these animals in the 200 mg/kg bw/day group were noted with macroscopic findings in the gastrointestinal tract (stomach, cecum, colon, duodenum, ileum, and/or jejunum was distended, eroded, thickened, and/or had dark red areas), indicating irritation from test substance administration. All other animals survived to the scheduled necropsies.

At the scheduled necropsy, test substance-related incidences of thickened stomach were noted for the 75 and 200 mg/kg bw/day group males and females and adhesions on the liver and spleen were noted for males in the 200 mg/kg bw/day group. All other macroscopic findings occurred in single animals and/or in a manner that was not dose-related.

The mean numbers of corpora lutea, unaccounted-for sites, and implantation sites in the 25, 75, and 200 mg/kg bw/day groups were similar to the control group values.

ORGAN WEIGHTS
Mean final body weight and organ weight alterations (i.e., adrenal glands and thymus) were considered to be associated with administration of the test substance.
Also, the mean brain weight relative to final body weight was significantly (p<0.05) higher in the 200 mg/kg bw/day group males. This difference was considered secondary to the lower final body weight in this group. No other differences were statistically significant when the test substance-treated groups were compared to the control group.

MICROSCOPIC EXAMINATIONS
Test substance-related histopathologic alterations in both males and females included hypertrophy of the zona fasciculata of the adrenal cortex and ulceration, epithelial hyperplasia and hyperkeratosis, and chronic-active inflammation in the non-glandular stomach. In the 200 mg/kg bw/day group males, there was also cytoplasmic vacuolation of the zona fasciculata of the adrenal cortex, and in the 200 mg/kg bw/day group females, there was lymphoid depletion in the thymus.

Ulceration was the focal, or typically multifocal, loss of the full-thickness of mucosal epithelium. Erosion, epithelial, was the focal or multifocal loss of a partial thickness of the mucosal epithelium. Erosion was not diagnosed when ulceration was present. Hyperplasia of the squamous epithelial mucosa of the non-glandular stomach was an increased thickness and irregular basal margin of the layers of the epithelium and it was invariably accompanied by hyperkeratosis, and increased thickness of the luminal layers of keratin which was not separately diagnosed. Chronic active inflammation was attributed to neutrophils as well as mononuclear inflammatory cells along with variably increased connective tissue. Inflammation was invariable at sites of ulceration but was not diagnosed separately unless it was also observed at other sites. Inflammation was invariable in the submucosa but also extended upward into the mucosa or downward into the gastric wall. In some instances, inflammation was transmural, extending throughout the gastric muscular wall to the outer serosa, and resulted in fibrous connective tissue adhesions of the gastric serosa to either the liver or the spleen. Hypertrophy of the adrenal cortex was an increase in the size of cells in the zona fasciculata. Cytoplasmic vacuolation of the adrenal cortex also occurred in the zona fasciculata. Lymphoid depletion in the thymus was a decreased number of lymphocytes in the cortex which made the distinction between the cortex and medulla less distinct.

There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No substance-related effects on reproductive performance at any dose

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

F1 LITTER DATA

PND 0 LITTER DATA AND POSTNATAL SURVIVAL
The mean number of pups born, live litter size, and the percentage of males at birth in the 25, 75, and 200 mg/kg bw/day groups were similar to the control group values. Postnatal survival in the 25, 75, and 200 mg/kg bw/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant.

GENERAL PHYSICAL CONDITION
The general physical condition of all F1 pups in this study were unaffected by test substance administration. Pups (litters) that were found dead numbered 15(4), 3(2), 4(3), and 4(4) in the control, 25, 75, and 200 mg/kg bw/day groups, respectively. Four (1), 0(0), 0(0), and 2(1) pups (litters) in these same respective groups were missing and presumed to have been cannibalized.

OFFSPRING BODY WEIGHTS
Mean male and female pup body weights and body weight changes in the 25, 75, and 200 mg/kg bw/day groups were unaffected by test substance administration during PND 1-4. Significantly (p<0.05 or p<0.01) higher mean male and female pup body weights were noted on PND 1 for the 75 mg/kg bw/day group. However, in the absence of a dose-related trend, these differences were not attributed to maternal test substance administration. No other statistically significant differences from the control group were noted. NECROPSIES OF PUPS FOUND DEAD

The numbers of pups (litters) found dead during PND 0-4 numbered 15(4), 3(2), 4(3), and 4(4) in the control, 25, 75, and 200 mg/kg bw/day groups, respectively. Aside from the presence or absence of milk in the stomach, no other internal findings were noted.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No substance-related effects on reproductive performance at any dose

Key result

Reproductive effects observed:

no

None

Conclusions:

Under the conditions of this screening study, no test substance-related effects were observed on reproductive performance at any dosage level. As such, a dosage level of 200 mg/kg bw/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of PETIA when administered orally by gavage to Crl:CD(SD) rats. 

Executive summary:

A study was conducted to determine the reproductive toxicity of PETIA to rats after oral exposure according to OECD Guideline 422.

Under the conditions of this screening study, no test substance-related effects were observed on reproductive performance at any dosage level. As such, a dosage level of 200 mg/kg bw/day was considered to be the NOAEL for reproductive toxicity of PETIA when administered orally by gavage to Crl:CD(SD) rats. 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A reproductive/developmental screening study was conducted in rats at 0, 25, 75 and 200 mg/kg bw/d (by gavage) to investigate potential adverse effect of PETIA on reproductive performance, including gonadal function, mating behavior, conception, parturition and early postnatal development, according to OECD Guideline 422. Under the conditions of this study, no test substance-related effects were observed on reproductive performance at any dosage level. Based on these results, 200 mg/kg bw/d was considered to be the NOAEL for reproductive toxicity. In the absence of any effects on the general physical condition of the F₁ pups, the NOAEL neonatal toxicity was 200 mg/kg bw/d (Stump, 2011). The above assessment of absence of reproductive effects is further supported by the absence of any evidence of toxicity of PETIA on the reproductive system in available repeated dose toxicity studies.

Effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 20, 2014 to September 02, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to standard guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

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: Charles River Laboratories France, L'Arbresle Cedex, France.
- Age at study initiation: 17-19 weeks
- Housing: individually housed in labelled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days prior to pairing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12-h light/12-h dark cycle

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations performed at WIL Research Europe
- Amount of vehicle (if gavage): 1 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (10 July 2014). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 h at room temperature under protection from light was also determined (highest and lowest concentration).

Duplicate samples (approximately 500 mg), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasks of 20 mL. For determination of accuracy, samples were taken at middle position (50% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the formulations. For determination of stability, additional samples were taken at 50% height and stored at room temperature protected from light for 6 h. The volumetric flasks were filled up to the mark with acetonitrile. The solutions were further diluted to obtain an end solution of 25/75 (v/v) acetonitrile/water and concentrations within the calibration range. All solutions containing the test substance were protected from light.

Analytical conditions:
- Instrument: Acquity UPLC system (Waters, Milford, MA, USA)
- Detector: Acquity UPLC TUV detector (Waters)
- Column: Acquity UPLC BEH Shield RP-18, 100 mm × 2.1 mm i.d., dp = 1.7 μm (Waters)
- Column temperature: 40°C±1°C
- Injection volume: 10 μL
- Mobile phase: 30/70 (v/v) acetonitrile/water
- Flow 0.45 mL/min
- UV detection: 225 nm


The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Details on mating procedure:

One female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was established by visual observation of mating. This day was designated Day 0 post-coitum.

Duration of treatment / exposure:

From Day 6 to Day 28 post-coitum, inclusive.
As a misgavage was suspected, female no. 45 (Group 3) was not dosed on Days 13 and 14 post-coitum. This animal died on Day 16 post-coitum.

Frequency of treatment:

Once daily for 7 days/week, approximately the same time each day
dose.

Duration of test:

From Day 6 to Day 28 post-coitum, inclusive

Remarks:

Doses / Concentrations:
0, 25, 50 and 75 mg/kg bw/day
Basis:

No. of animals per sex per dose:

24, 22, 26 and 24 females in groups 1, 2, 3 and 4, respectively; group 1 being control

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected based on the results of a dose range finding study. In this study, six mated rabbits per groups were dosed at 0, 25, 50 or 75 mg/kg bw/day for Days 6 to 28 postcoitum,inclusive, by oral gavage. At 75 mg/kg bw/day, toxicity consisted of reduced faeces production, reduced body weight gain (with a body weight loss on Days 6 to 9 post-coitum) and reduced food consumption on Days 6 to 9 post-coitum. At 75 mg/kg bw/day, one female had dark red fluid in the uterus. This animal had 5 live fetuses and 6 early resorptions, which resulted in a slightly increased post-implantation loss at this dose. At all dose levels, fetal body weights were slightly lower compared to controls, but this was not statistically significant and did not show a dose relationship.

Maternal examinations:

MORTALITY/VIABILITY:
At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ (2000)7). The circumstance of any death was recorded in detail.

CLINICAL SIGNS:
At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) were scored. Cage debris was examined in case of abortions.

BODY WEIGHTS:
Days 0, 3, 6, 9, 13, 16, 20, 23, 26, 29 post-coitum.

FOOD CONSUMPTION:
Days 0-3, 3-6, 6-9, 9-13, 13-16, 16-20, 20-23, 23-26 and 26-29 post-coitum.

WATER CONSUMPTION:
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

POST-MORTEM EXAMINATIONS:
All animals surviving to the end of the observation period, all moribund animals and all animals showing abortion were euthanized and subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). Females which had abortion or spontaneous death, were necropsied within 24 h of abortion.

Ovaries and uterine content:

Each ovary and uterine horn of animals surviving to planned necropsy was dissected and examined as quickly as possible to determine:

- The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The weight of each fetus.
- The sex of each fetus.
- Externally visible macroscopic fetal abnormalities.

Animals found dead or sacrificed before planned necropsy, were subjected to relevant examinations of the ovaries and uterine horns.

Fetal examinations:

External, visceral, and skeletal findings were recorded as developmental variations or malformations.

External:
Each viable fetus was examined in detail and weighed. All live fetuses were euthanized. The nonviable fetus (the degree of autolysis was minimal or absent) was examined, crown-rump length measured and weighed. The crown-rump length of late resorptions was measured and a gross external examination performed. Late resorptions with malformations were fixed in 10% buffered formalin.

Visceral (Internal):
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. The sex of all fetuses was determined by internal examination. The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution. Tissues were then transferred to a 70% aqueous ethanol for subsequent processing and soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues were stored in 10% formalin. All carcasses, including the carcasses without heads, were eviscerated, skinned and fixed in identified containers containing 96% aqueous ethanol for subsequent examination of skeletons.

Skeletal:
The eviscerated fetuses from Groups 1 and 4, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S. Subsequently, the skeletal examination was done on all fetuses from Groups 1 and 4. Since no treatment related effects in the high dose group were seen, skeletal examination was not extended to the fetuses from the low and mid dose group. All specimens were archived in glycerin with bronopol as preservative. A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. Evaluation by the fetal pathologist and study director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY:
No toxicologically relevant mortalities occurred up to 75 mg/kg bw/day.
Ten animals died preterm
-Five animals showed an abortion and had to be terminated early. Macroscopic examination showed abnormalities of the heart (enlarged, pale or soft), stomach (many black foci on glandular mucosa, many black-brown or dark red foci, irregular surface of the forestomach or gelatinous contents), gallbladder (enlarged or many greenish foci), oviducts (several watery-clear cysts), pancreatic lymph nodes (enlarged and dark red), alopecia, thoracic cavity (containing watery-clear fluid), lungs (many, black foci), liver (pale), general pale discolouration, or watery-clear contents in the caecum.
- Five animals died due to complications of the gavage procedure. Macroscopic examination of these animals revealed abnormalities in the trachea (contents: reddish/dark red foamy or hemorrhagic/clotted blood), lungs (reddish foamy contents, many dark red foci, dark red discolouration or isolated tan focus), or advanced autolysis.

CLINICAL SIGNS
No toxicologically relevant clinical signs were noted up to 75 mg/kg bw/day. Incidental findings that were noted included alopecia, scars, scales, scabs, lean appearance, hunched posture, laboured respiration, rales, ulcer, diarrhoea, a wound, and salivation. These findings occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

BODY WEIGHTS
At 50 and 75 mg/kg bw/day, overall a body weight loss was noted on Day 9 post-coitum and reduced body weight gain was noted on Day 13 post-coitum. These changes were statistically significant but did not show a dose response. During the remainder of treatment, mean body weights recovered to normal. Corrected (for uterus) body weight gain was unaffected at these dose levels. At 25 mg/kg bw/day, body weights and (corrected) body weight gain remained in the same range as controls over the treatment period.


FOOD CONSUMPTION
Absolute and relative food consumption were statistically significantly lower on Day 6-13 post-coitum at 75 mg/kg bw/day and on Day 6-9 post-coitum at 50 mg/kg bw/day. Food consumption was slightly lower on Day 13-16 post coitum (not statistically significant). Subsequently, mean food consumption recovered to normal. Food consumption before or after allowance for body weight of treated animals at 25 mg/kg bw/day remained in the same range as controls.

WATER CONSUMPTION
During the observation period several animals in all groups (vehicle control and test item treated) showed reduced water intake on one or more days. In the absence of a clear treatment-related effect, no quantitative measurement of water consumption was introduced during this study.

MACROSCOPIC EXAMINATION
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. The incidence of incidental findings among control and treated animals was within the background range of findings that are encountered among rabbits of this age and strain, and did not show a dose-related trend. These necropsy findings were therefore considered to be of no toxicological relevance.

MATERNAL PREGNANCY DATA
For the control, low, mid and high-dose groups, there were respectively 22, 21, 21 and 19 litters with viable fetuses available on Day 29 post-coitum. In the control group, out of 24 mated animals two died preterm; all rabbits were pregnant. At 25 mg/kg bw/day, out of 22 mated animals one had an abortion. At 50 mg/kg bw/day, out of 26 mated animals two aborted, two died preterm (both pregnant) and one was not pregnant. At 75 mg/kg bw/day, out of 24 mated animals two aborted, one died preterm (pregnant) and two were not pregnant.

Key result

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
LITTER SIZE
Litter sizes were not affected by treatment up to 75 mg/kg bw/day. Mean litter sizes were 9.5, 8.6, 9.2 and 8.4 viable fetuses per group for the control, 25, 50 and 75 mg/kg bw/day group, respectively. There was no effect on fetal viability up to 75 mg/kg bw/day. Litter proportions of viable or dead fetuses and early resorptions were all within the normal range of biological variation. The percentage of late resorptions at 25 mg/kg bw/day was slightly outside the historical control data (4.0% versus a maximum of 3.5% in the historical control data). As no dose response was noted, this slight increase was not considered toxicologically relevant.

SEX RATIO
Sex ratio was unaffected by treatment up to 75 mg/kg bw/day.

FETAL BODY WEIGHT
Fetal body weight was unaffected by treatment up to 75 mg/kg bw/day. Mean fetal body weights (both sexes combined) were 36.3, 37.8, 35.5 and 37.5 grams in the control, 25, 50 and 75 mg/kg bw/day group, respectively.

FETAL MORPHOLOGICAL EXAMINATIONS
The numbers of fetuses (litters) available for external and visceral morphological evaluation were 208 (22), 180 (21), 194 (21) and 160 (19) in control, 25, 50 and 75 mg/kg bw/day groups respectively. Soft tissue cephalic examination was done for approximately half of the fetuses of all groups. Skeletal examinations were performed for all fetuses of control and 75 mg/kg bw/day groups.

EXTERNAL MALFORMATIONS AND VARIATIONS
There were no external developmental malformations or variations up to 75 mg/kg bw/day for fetuses at planned necropsy. Two fetuses of one litter from 50 mg/kg bw/day group showed hyperextension of the right hind limb. As this only affected one litter and this malformation was noted in the historical control data, it was not considered toxicologically relevant.

VISCERAL MALFORMATIONS AND VARIATIONS
There were no treatment related effects on visceral morphology following treatment up to 75 mg/kg bw/day. Retrocaval ureter was noted at 0.8% in the control group, 1.8% at 25 mg/kg bw/day, 1.1% at 50 mg/kg bw/day and 2.6% at 75 mg/kg bw/day. At 25 and 75 mg/kg bw/day, this was just outside the historical control maximum of 1.4%. Without a clear dose response relationship, these slight increases were not considered toxicologically significant. Any remaining visceral malformations and variations were not considered treatment related as they occurred infrequently, did not follow a dose-related trend, or occurred at frequencies that were within the range of available historical control data.

SKELETAL MALFORMATIONS AND VARIATIONS
At 75 mg/kg bw/day, there was a slight increase in skeletal developmental variations. This included three parameters indicating a developmental delay: unossified metacarpals and/or metatarsals (11.7% at 75 mg/kg bw/day versus 7.7% in the control group), slightly or moderately mal-aligned sternebrae (6.0% at 75 mg/kg bw/day versus 1.8% in the control group) and unossified tarsals (2.9% at 75 mg/kg bw/day versus 1.1% in the control group). As these increases were only slight and not statistically significant, these were not considered toxicologically significant. Additionally, the number of fetuses with caudal shift of the pelvic girdle was increased at 75 mg/kg bw/day (31.7% per litter; 52 fetuses in 11 litters) compared to the control group (17.1% per litter; 36 fetuses in 13 litters). Without any corroborative findings and as the number of affected litters was even less than controls, this finding was not considered toxicologically relevant. All malformations and remaining skeletal variations recorded in this study were not considered to be treatment related as they occurred infrequently, at lower levels in the high dose group, occurred at frequencies that were within the range of available historical control data, or were noted in control fetuses only.

Key result

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect was observed at the highest dose tested

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

None

Conclusions:

Under the conditions of the study, the No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity for the test substance was established as being at least 75 mg/kg bw/day, since no adverse effect was observed.

Executive summary:

A prenatal developmental toxicity study was conducted with PETIA in rabbits after oral exposure according to OECD Guideline 414, EU method B.31 and EPA OPPTS 870.3700 in compliance with GLP.

Ninety six mated female rabbits were assigned to four dose groups. The number of animals was 24, 22, 26 and 24 for Groups 1, 2, 3 and 4, respectively. The test substance was administered once daily by oral gavage from Days 6 to 28 post-coitum, inclusive, at doses of 25, 50 and 75 mg/kg bw/day (Groups 2, 3 and 4, respectively). The rabbits of the control group received the vehicle, Arachid oil, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations.

No maternal or developmental toxicity was observed in any of the groups. Under the conditions of the study, the No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity for the test substance was established as being at least 75 mg/kg bw/day, since no adverse effect was observed.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A reproductive/developmental screening study was conducted in rats at 0, 25, 75 and 200 mg/kg bw/d (by gavage) to investigate potential adverse effect of PETIA on reproductive performance, including gonadal function, mating behavior, conception, parturition and early postnatal development, according to OECD Guideline 422. Under the conditions of this study, no test substance-related effects were observed on reproductive performance at any dosage level. Based on these results,200 mg/kg bw/dwas considered to be theNOAEL for reproductive toxicity. In the absence of any effects on the general physical condition of the F₁ pups, the NOAEL neonatal toxicitywas200 mg/kg bw/d (Stump, 2011).

A prenatal developmental toxicity study was conducted with PETIA in rabbits after oral exposure according to OECD Guideline 414, EU method B.31 and EPA OPPTS 870.3700, in compliance with GLP. Ninety six mated female rabbits were assigned to four dose groups. The number of animals was 24, 22, 26 and 24 for Groups 1, 2, 3 and 4, respectively. The test substance was administered once daily by oral gavage from Days 6 to 28post-coitum, inclusive, at doses of 25, 50 and 75 mg/kg bw/day (Groups 2, 3 and 4, respectively). The rabbits of the control group received the vehicle, arachid oil, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. All animals surviving to Day 29post-coitumwere subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations andcorpora luteawere recorded. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. Neither maternal nor developmental toxicity was observed in any of the groups. Under the conditions of the study, the NOAEL for maternal and developmental toxicity for the test substance was established as being at least 75 mg/kg bw/day (Beekhuijzen, 2014).

A study was conducted to evaluate the embryo/fetal toxicity and teratogenic effects of C-253 when administered to a group of 22 pregnant female rats. The test substance was administered at a single dose level of 100 mg/kg bw/day to pregnant rats from Gestation Day (GD) 6-15. All animals were observed once daily and records made of mortality, moribundity and clinical signs. Body weights were recorded. On Day 20 of gestation, females were sacrificed by carbon dioxide asphyxiation and the fetuses were taken by caesarean section. Maternal, ovarian, uterine, litter and fetal data were evaluated. Test substance related maternal toxicity consisting of decreased survival, decreased body weight gains, or clinical signs (wheezing, dyspnea, rough haircoat, hunched posture, bloody crust around eyes, nose, mouth, and forepaws, salivation, and alopecia) were observed. Fetotoxic effects (probably related to maternal toxicity) were observed and consisted of an increase in resorptions and decreases in percent fetal viability, mean fetal weight, and mean gravid uterine weight. Under the conditions of the study, maternal toxicity and fetotoxicity was observed for test substance, when administered to pregnant rats at a level of 100 mg/kg bw/day. The fetotoxic effect was probably resulted from maternal toxicity. Treatment was terminated at the request of the sponsor due to excessive mortality at the dose administered (Serota, 1982; Serota, 1983).

A study was conducted to evaluate the embryo/fetal toxicity and teratogenic effects of C-253 when administered to a group of 25 pregnant rats. The test substance was administered at a single dose level of 10 mg/kg bw/day to pregnant rats from Gestation Day (GD) 6-15. All animals were observed once daily and records made of mortality, moribundity, and clinical signs. Body weights were recorded. On Day 20 of gestation, females were sacrificed by carbon dioxide asphyxiation and the fetuses were taken by caesarean section. Maternal, ovarian, uterine, litter and fetal data were evaluated. Suggestive signs of maternal toxicity were noted with the death of one female and the observation of wheezing in several animals. No effects, however, were noted in body weights, food and water consumption values, or gross pathology. An increased incidence of skeletal variants above the normal expected incidence was noted in the fetuses examined and consisted primarily of lagging ossification of various skeletal elements. This effect was considered treatment related but whether it resulted from maternal toxicity or from a direct fetotoxic effect could not be established. Under the conditions of the study, probable maternal toxicity and/or fetotoxicity was observed for test substance, when administered to pregnant rats at a level of 10 mg/kg bw/day. The fetotoxic effect was considered treatment related but whether it resulted from maternal toxicity or from a direct fetotoxic effect could not be established. No evidence of a teratogenic effect was noted (Serota, 1984).

The available guideline compliant developmental toxicity and teratology screening studies for PETIA in rabbits and rats support the conclusion that the substance does not represent a developmental toxicity hazard in humans. Particularly, the recent OECD Guideline 414 developmental toxicity study in rabbits as well as the OECD Guideline 422 reproductive/developmental screening study in rats did not reveal any evidence of developmental toxicity. These findings are supported by absence of developmental effects of PETIA at maternally non-toxic doses in a series of teratology studies in rats (Serota, 1982; Serota, 1983; Serota, 1984). While the main teratology screening study with PETIA (Serota, 1984) was only rated as Klimisch 3 due to the absence of a control group, this study belonged to a bigger teratology testing programme for a series of acrylates and has to be seen in context of the Serota study of 1983. Initially, PETIA was considered in the Serota study of 1983 which included a control group, but was then excluded due to a too high level of toxicity. The teratology testing of PETIA was then repeated shortly after at a lower dose level (Serota, 1984). It is of note that both studies were conducted in sequence at the same reputable laboratory (i.e., Hazleton). In none of the reports was any statement made indicating that the control groups were outside the historical control values of the specific laboratory. This gives an additional degree of confidence to consider the information provided in the teratology screening with PETIA (Serota, 1984), even in the absence of a control group.

In conclusion, in addition to the available evidence of absence of available toxicity from the OECD Guideline 414 developmental toxicity study in rabbits, the weight of the evidence suggests that PETIA does not cause developmental toxicity in rats.

Justification for classification or non-classification

A reproductive/developmental screening study was conducted in rats to investigate potential adverse effect of PETIA on reproduction, including embryo/foetal development. No adverse effects on reproductive/developmental parameters were noted at doses up to 200 mg/kg bw/d in a repeated dose and reprotox screening study. Also, there were no effects on the general physical condition of the F1 pups at any dose. The substance therefore does not qualify for classification for these endpoints. A developmental toxicity study conducted with rabbits has not led to adverse effects up to the highest dose level.