N-tert-butylacrylamide

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on developmental toxicity

Description of key information

Study 1

In the OECD 414 study conducted with Wistar rats, the NOAELs for maternal developmental toxicity and foetal developmental toxicity were considered at 500 and 125 mg/kg/day, respectively. NOAEL for maternal general toxicity could not be established due to significant decreases in body weight gain and food intake at 125 mg/kg/day. The fetal effects observed at 250 and 500 mg/kg/day were limited to decreased fetal weights (by about 11% at 250 mg/kg/day and by about 13% at 500 mg/kg/day). These fetal effects were considered secondary to maternal toxicity, which at 250 and 500 mg/kg/day included significant decreases in body weight gain during different periods of gestation e.g. GD 5-20 (by 19% at 250 mg/kg, and by 29% at 500 mg/kg), significant decreases in absolute body weight (by 5-10% at 250 mg/kg/day, and by 7-13% at 500 mg/kg/day), and significant reductions in food intake during different periods of gestation (by 9-27% at 250 mg/kg/day, and by 16-49% at 500 mg/kg).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

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:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Species and strain : Wistar Rats
Source : Hylasco Biotechnology (India) Pvt. Ltd.4B, MN Park, Shameerpet Mandal,Turkapally Village, Medchal District, Telangana -500078
Age at the start of treatment : 12 weeks
Mean body weight, SD and body weight range of Day 0 mated females: The weight variation of rats was minimal and did not exceed ± 20 % of the mean body weight in any group. Mean body weight (g) Bwt. range (g) G1: 250.939 ± 16.558 (range: 221.98 to 278.92) G2: 254.124 ± 16.324 (range: 224.82 to 284.80) G3: 252.671 ± 18.292 (range: 209.92 to 284.61) G4: 254.128 ± 15.750 (range: 219.54 to 284.03).

Acclimatization : After detailed clinical examination for good health and suitability for the study, 120 females and 50 male rats were acclimatized
for five days before initiation of mating. Only nulliparous and non-pregnant females were used in the study

Environmental conditions: Rats were housed under standard laboratory conditions, air conditioned with adequate fresh air supply (12 to 15 air changes/hour). Environment: with temperature 20 to 22 °C, relative humidity 65 - 66 %, with a 12 hours light and 12 hours dark cycle. The maximum and minimum temperatures in the experimental room were recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.

Housing: Rats were housed in standard polysulfone rat cages (size: Length 425 mm × Width 266 mm × Height 185 mm) with stainless steel top grill having facilities for pellet food and drinking water in polycarbonate bottle with stainless steel sipper tube. Additionally, polycarbonate rat huts were placed inside the cage as environmental enrichment objects which were replaced once a week.
i. Pre-mating / Acclimatization: Two rats of the same sex per cage were housed.
ii. Mating: Female rats were cohabited with males in a 1:1 ratio in the same cage.
iii. Post-mating / Treatment: After mating confirmation, females were housed individually. Nesting material (paper cuttings) were placed inside the cages from GD 18 onwards.

Bedding: Steam sterilized corn cob was used as bedding material and was changed along with the cage at least twice a week.

Diet and water: Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, was available ad libitum to the rats. Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India was available ad libitum to rats in
polycarbonate bottles with stainless steel sipper tubes. The food and water provided to the rats were tested for contaminants.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Milli-Q water

Details on exposure:

Vehicle alone and test item formulations were administered by oral gavage using a disposable plastic syringe attached with a metal feeding cannula to rats of specific groups daily from GD 5 to GD 19 of presumed gestation, at approximately the same time each day (varying by ± 2 hours). The test item was administered at a fixed dose volume of 10 mL/kg body weight. The actual volume was calculated for individual animals using the most recently recorded body weight. Homogeneity of the test item suspensions during sampling was maintained by constant stirring using magenetic stirrer. The animals in the vehicle control group were handled in an identical manner to the treatment group and were administered vehicle (Milli-Q water) only.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for analysis of homogeneity and active ingredient were collected from the dose formulations intended for first treatment for the first batch of rats and at 1-2 days before termination of treatment. The prepared formulations were sampled in duplicate sets wherein one set was used for analysis and another as backup set which was stored at ambient condition. For each set, duplicate samples were drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from middles layer was drawn. The collected samples were sent to Analytical R&D of Eurofins Advinus Ltd. for formulation analysis to determine the concentration of the dose formulation. The analysis was carried out as per the method validated under Eurofins Advinus Study No.: G20360. The results obtained for G4 group samples at initiation of treatment (12.10.2020) were found to be out of acceptance limit, hence analysis was repeated using fresh formulation prepared next day (13.10.2020) Formulations were considered acceptable as the mean results were within ± 15.0 % of the nominal concentration and the relative standard deviation (% RSD) was less than 10.0 %.

Details on mating procedure:

During the mating period, female rats were cohabited with males in a 1:1 ratio and vaginal smears and / or vaginal plug were examined in the morning hours of the subsequent day. If sperm was detected in a vaginal smear and/or if a vaginal plug was observed in the morning, the female was considered to be mated. This day was regarded as GD 0. The females were cohabited in batches of required numbers. This procedure was continued until there were sufficient numbers of GD 0 pregnant rats for the study. GD 0 pregnant rats obtained on each day were randomly distributed to different groups by body weight stratification method using ProvantisTM software. After grouping and ascertaining the group mean body weight, the rats were allocated accession number as applicable to the group on each day of assignment. Note: After confirming mating, females were housed individually, and the unselected females were euthanized. The males were returned back to stock after mating procedure. The selected female rats were assigned to vehicle control and treatment groups.

Duration of treatment / exposure:

GD 5 to 19 (15 days).

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

G2

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

G3

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

G4

No. of animals per sex per dose:

24 female rats per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The study was designed to use minimum number of animals to meet the scientific objectives, the goals of the Sponsor and considering applicable
regulatory requirements. This study was performed in an AAALAC approved laboratory. All procedures were in compliance with the guidelines issued by the Committee for Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. This study plan has been approved by the Institutional Animal Ethics Committee (IAEC) of Eurofins Advinus Limited (Approval No.: 010/ June 2020).

Maternal examinations:

Observations for clinical signs were performed twice per day during treatment (at approx. 30 minutes pre-dosing and at 1 to 2 hours post-dosing) and at least once per day during treatment-free days. Each rat was observed twice daily during treatment period for morbidity and mortality i.e., once in the morning and once in the afternoon. Based on the lack of clinical signs, the observation for morbidity and mortality was carried out
once during weekends and public holidays.
All females included in the study were weighed on gestation days 0, 3, 5, 8, 11, 14, 17 and 20. Intermittent body weight gains were calculated from GD 0–3, 3–5, 5–8, 8–11, 11–14, 14–17 and 17–20. Further body weight gains for pre-treatment period (GD 0–5), treatment period (GD 5–19), and for entire gestation period (GD 0–20) were derived and analyzed only for rats pregnant at caesarean section. The corrected body weight was obtained by subtracting the unopened uterine weight from terminal body weight (body weight on GD 20). The corrected body weight gain was calculated by subtracting the body weight on GD 5 from the corrected body weight.
About 200 g of food (food input) was provided on GD 0. The food output was recorded and replenished to about 200 g on GD 3, 5, 8, 11, 14 and 17. Prior to terminal sacrifice, food left over was recorded on GD 20. The quantity of food consumed by each female was calculated for the
following intervals: gestation days GD 0–3, 3–5, 5–8, 8–11, 11–14, 14–17 and 17–20. Food consumptions for pre-treatment period (GD 0-5), treatment period (GD 5-19) and for entire gestation period (GD 0-20) were also derived. Food consumptions were statistically analyzed and presented only for rats found pregnant at caesarean section. No spillage of food was observed during the food measurement intervals.

At caesarean section, a blood sample was collected from each rat under isoflurane anaesthesia, with a fine capillary tube, by retro-orbital sinus puncture for the determination of total Triiodothyronine (T3), Thyroxine (T4) and Thyroid Stimulating Hormone (TSH) hormone levels. Blood samples (about 1 mL from each rat) were collected in plain labeled tubes and kept on bench top for 90 minutes before centrifugation. Serum was
separated by centrifuging the whole blood samples at 5000 rpm for 5 minutes at 4°C. The serum samples were placed in labeled plastic tubes and stored at below -70°C until they were analysed. The left-over samples from hormone analysis were discarded on the day of data review at the discretion of Study Director/Study Pathologist.

Ovaries and uterine content:

Prior to caesarean section, code numbers were generated for blindfolding the animal numbers in order to avoid bias during caesarean section and subsequent fetal evaluation. The animal code was written on the tail by striking out the permanent accession number. On GD 20, all rats were sacrificed (caesarean section) under isoflurane anaesthesia and gross pathological changes in placenta and in all visceral organs of rats were examined. Gross necropsy involved external observation and examination of thoracic and abdominal viscera including uterine contents which were performed on all rats in the study sacrificed at term (caesarean section). At necropsy, the thyroid gland was collected and weighed from each rat (post-fixation) and subjected to histopathological examination. The tissue was processed for routine paraffin embedding and 4-5-micron sections were stained with Haematoxylin and Eosin stain. Remaining non-used tissues were archived. Immediately after termination, the gravid uterus along with the cervix wasexcised, weighed, and immediately examined. The following maternal endpoints were investigated:
a. Pregnancy status
b. Gravid uterine weight
c. Number of corpora lutea
d. Number of implantation sites
e. Number of early resorptions
f. Number of late resorptions
g. Gross evaluation of placenta
Uteri from rats that appeared non-gravid were subjected to 10% ammonium sulphide staining to observe implantation sites if any (identified as pregnantrats) or to confirm the non-pregnant status.

Fetal examinations:

On GD 20, the individual fetuses were delivered by hysterectomy and removed sequentially. The fetuses were euthanized using intraperitoneal injection of sodium thiopentone. Fetuses were subjected to external and visceral or skeletal examination.
The following litter endpoints were investigated:
a. Total number of fetuses
b. Number of live/dead fetuses
c. Individual fetal body weight (g)
d. Anogenital distance (mm) from all live fetuses
e. Sex of the fetus - external determination based on anogenital distance that was confirmed based on gonadal examination (internal sex) during
visceral examination

Fetuses were examined for external and visceral or skeletal alterations and the observations were classified as variants, anomalies and malformations as defined below:
Variants: An incidence distributed throughout a population in consistently large percentage including the ossification variations.
Anomaly: Alteration in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal.
Malformation: Structural anomalies that alter general body conformity, disrupt or interfere with normal body function, may have a detrimental impact on postnatal life, or may be incompatible with postnatal survival. All fetuses were examined for external alterations Approximately one half the number of fetuses from each litter were subjected to visceral evaluation and the remaining half the number of the fetuses were
subjected to skeletal evaluation.
Fetal Visceral Evaluation: Fetuses from each litter were prepared for fresh tissue visceral organ evaluation. The fetuses subjected for visceral evaluation were decapitated and heads were stored in 70% alcohol in an individual bottles labeled with study number, code number and fetal number among total number of fetuses. Further heads were examined by sectioning using modified Wilsons Razor blade sectioning technique. After head evaluation, as no abnormalities were observed, fetal heads were discarded.
Fetal Skeletal Evaluation: The remaining half of the fetuses of each litter were prepared for skeletal evaluation by wet skinning followed by evisceration and staining. Fetuses were fixed in 70 % ethyl alcohol, eviscerated, and dehydrated in 95 % ethyl alcohol; macerated in 1.5 % KOH and stained with saturated, aqueous Alizarin red S in Mall’s solution. The excess stain was removed in Mall’s solution and fetuses were cleared by passing through grades of glycerol with thymol crystals. Individual bottles containing fetuses for skeletal evaluation were labeled with study number, code number and fetal number among total number of fetuses. After the skeletal evaluation, fetuses were pooled dam-wise in bottles containing 100 % glycerol with crystals of thymol to prevent fungal growth. The bottles were labeled with study number, code number and number of fetuses evaluated among total number of fetuses. All findings recorded are presented in the report as per the standard reporting format. The malformation observed during fetus skeletal evaluations were cross verified by another specialist in prenatal developmental toxicology study.

Statistics:

The data on maternal body weight, body weight change in interval, gravid uterine weight, body weight change corrected to gravid uterine weight,
maternal food consumption, hormone analyses (T4, T3, TSH), and the weight of thyroid gland were analyzed using Analysis of Variance (ANOVA) after
testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after
suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group
differences were found significant.Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate forgroup. Anogenital distance for male and female was analyzed using Analysis of Covariance (ANCOVA) taking weight as covariate for group. Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss observations were analyzed using Kruskal-Wallis test for group comparison. Wilcoxon pairwise comparisons of the treated groups with the control group were performed, when the group differences were significant. The incidence of dams with resorptions were tested for using Chi-square test followed by Fisher’s exact test for group association. The incidence of fetus and litter (incidence and percent) observations for external, visceral and skeletal observations were tested using Cochran-Armitage trend test and pair-wise comparison will be tested by Fisher’s exact test for group association. Statistically significant differences (defined as p<0.05) in the aforementioned analyses are designated as * throughout the report.

Historical control data:

In-house historical control data was taken into account during data interpretation.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs attributed to the Test Item administration.

Mortality:

no mortality observed

Description (incidence):

All animals survived to planned death.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant decreases in maternal body weight were observed at 250 mg/kg (On GD 11, 14, 17 and 20) and 500 mg/kg (On GD 8, 11, 14, 17 and 20). The decreases at 250 mg/kg were in the range of 5 to 10% whereas the decreases at 500 mg/kg were in the range of 7 to 13%. No significant decrease in maternal body weight were observed at 125 mg/kg. In terms of maternal body weight gain, significant decreases were observed from GD 5 to 20 at 125 mg/kg (by 18.7%), 250 mg/kg (by 29.5%) and at 500 mg/kg (by 41.3%)
The corrected mean body weights in all treated groups were significantly lower(by 5 to 12%) when compared to the control group. Corrected mean body weight gains in all treated groups were also significantly lower (by 30 to 82%)when compared to the control group.
The effects on body weight gain at 125, 250 and 500 mg/kg/day were attributed to the test item.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

As compared to the control group, significant decreases in food intake were observed at 250 and 500 mg/kg/day, during GD 5 - 8, 8 - 11, 11 - 14, 14 – 17 and 17 – 20 and significant decreases at 125 mg/kg/day was observed during GD 5-8, 11-14 and 17 to 20. The decreases ranged from 6 to12% at 125 mg/kg/day, from 9 to 27% at 250 mg/kg/day and from 16 to 49% at 500 mg/kg/day. Consequently, net food intakes at 125, 250 and 500 mg/kg during treatment (GD 5 - 20) and the entire gestation period (GD 0-20) were significantly lower compared to the control group.The effects on food intake at 125, 250 and 500 mg/kg/day were attributed to the test item.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No significant changes in T3, T4, or TSH levels were observed at any dose level.

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:

effects observed, treatment-related

Description (incidence and severity):

Significant decreases in uterine weight were observed at 250 mg/kg (by 20%) and 500 mg/kg (by 19%) compared to the control. No significant changes in thyroid gland weight were observed at any dose level.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes in any animal at any dose level that could be attributed to the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid: Ultimobranchial cysts were observed in 2, 1, 0, and 1 dam treated at 0, 125, 250 and 500 mg/kg/day, respectively. These cysts lacked dose dependency and were considered incidental in nature and therefore unrelated to test item administration. No other histopathological findings in the thyroid were observed in any of the dams at any dose level.

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Number of rats non-pregnant at caesarean section were 4, 4, 2, and 1 at 0, 125, 250 and 500 mg/kg bw/day.

Other effects:

no effects observed

Description (incidence and severity):

No significant changes in the number of corpora lutea.

Dose descriptor:

NOAEL

Remarks:

General toxicity

Based on:

test mat.

Remarks on result:

not determinable

Dose descriptor:

LOAEL

Remarks:

General toxicity

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Dose descriptor:

NOAEL

Remarks:

maternal developmental toxicity

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

changes in number of pregnant

dead fetuses

early or late resorptions

maternal abnormalities

necropsy findings

number of abortions

pre and post implantation loss

total litter losses by resorption

other: Number of corpora lutea

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant decreases in male fetal weight, female fetal weight, and sex-combined fetal weight were observed at 250 mg/kg (by approx. 11% vs control for all cases) and 500 mg/kg (by approx. 13% vs control for all cases).

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

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One small fetus (defined as anomaly) out of a total of 293 fetuses was observed at 0 mg/kg/day. No other external findings were observed at 0, 125, 250 or 500 mg/kg/day.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no skeletal malformations observed in any litter at any dose level.

The following variations were observed: significant increases of incomplete ossification of skull bones (parietal, interparital, supraoccipital and hyoid bone)
at 250 and mg/kg; significant increase of delayed ossification of cervical vertebral center no. 1 at 250 mg/kg; significant increase of delayed ossification
of sternum no. 6 at 500 mg/kg; significant increase of incomplete ossification of sternum no. 6 at 250 mg/kg; significant increases of incomplete ossification
of sternum no. 4 at 250 and 500 mg/kg; significant increases of incomplete ossification of sternum no. 3 at 125, 250 and 500 mg/kg; and a significant
increase of incomplete ossification sternum no. 1 at 500 mg/kg. Delayed/incomplete ossifications are generally considered transient changes and these finding denote generalized growth delays which normally ossify late in gestation. They also do not have general predictive value for teratogenicity [Carney and Kimmel (2007)]. Therefore, these variations were considered to be non-adverse.

The following skeletal anomalies were observed: significant increases of hypoplstic sternum no. 2 at 125, 250 and 500 mg/kg; and a significant increase
of dumb-bell shaped thoracic centrum no. 9 at 500 mg/kg/day. These anomalies in these anatomic structures were considered to have no significant
biological impact on animal health or body conformity, hence; they were not considered to be adverse effects of treatment.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed during visceral examination at 125, 250 or 500 mg/kg/day. There was one incidence of renal pelvis dilation (anomaly; moderate in severity) and one incidence of ureter dilatation (anomaly; moderate in severity) at 500 mg/kg/day (one each out of 150 fetuses). There was also one incidence of heart innominate artery absent (malformation) at 0 mg/kg/day (1/142 fetuses). These mentioned incidences were considered spontaneous in nature and were therefore not attributed to the test item. Fetal heads in all dose groups were normal when subjected to Wilsons-Razor blade sectioning.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

A small, but statistically significant decrease in male AGD (by less than 5%) was observed at 500 mg/kg. This effect was not considered to be an adverse effect of treatment since mean AGD in male rats treated at 500 mg/kg bw/day (mean 2.43 mm) was within the historical control range (i.e. within 2.22 to 3.41 mm). No other significant changes in litter data were observed at 250 or 500 mg/kg.

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

other: AGD and AGD ratio

Dose descriptor:

LOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Abnormalities:

no effects observed

Developmental effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Table 1. Summary of Maternal Survival, Pregnancy Status and Fetus Disposition

Parameters	Group No.	G1	G2	G3	G4
	Dose (mg/kg/day)	0	125	250	500
Total No. of rats found sperm positive / group		24	24	24	24
Duration of treatment		GD 5 to 19 (total 15 days)
Caesarean section (day of presumed gestation)		GD 20
Number of rats sacrificed at caesarean section		24	24	24	24
Number. of rats non-pregnant at caesarean section		4	4	2	1
Number of rats pregnant and litters examined at caesarean section		20	20	22	23
Total number of fetuses		293	267	287	314
Total number of dead fetuses		0	0	0	0
Number of fetuses evaluated
a. External examination		293	267	287	314
b. Visceral examination		142	129	138	150
c. Skeletal examination		151	138	149	164

Table 2. Summary of Clinical Signs and Mortality

Observation Type: All Types	Female
	G1 0 mg/kg/day	G2 125 mg/kg/day	G3 250 mg/kg/day	G4 500 mg/kg/day
Normal Killed ‑ terminal kill	24	24	24	24
	24	24	24	24

Table 3. Summary of Maternal Body Weight of Pregnant Rats

Sex: Female			G1 0 mg/kg/day	G2 125 mg/kg/day	G3 250 mg/kg/day	G4 500 mg/kg/day
Body Weight (g)	0 [a]	Mean SD N %Diff	250.29 12.89 20 -	250.49 14.13 20 0.08	251.82 13.06 22 0.61	250.95 10.80 23 0.27
	3 [a]	Mean SD N %Diff	269.37 14.71 20 -	268.37 14.32 20 -0.37	269.54 14.10 22 0.06	270.53 10.71 23 0.43
	5 [a]	Mean SD N %Diff	280.52 15.65 20 -	279.18 14.07 20 -0.48	280.08 13.82 22 -0.16	280.77 10.51 23 0.09
	8 [a]	Mean SD N %Diff	291.40 17.66 20 -	283.75 13.74 20 -2.62	281.13 13.14 22 -3.52	270.23* 11.63 23 ‑7.26
	11 [a1]	Mean SD N %Diff	310.09 19.42 20 -	300.85 15.24 20 ‑2.98	296.06* 13.51 22 ‑4.53	284.74* 9.4 23 ‑8.17
	14 [a2]	Mean SD N %Diff	328.18 22.63 20 -	315.65 19.16 20 ‑3.82	308.43* 12.76 22 ‑6.02	298.47* 10.34 23 ‑9.05
	17 [a1]	Mean SD N %Diff	361.17 27.54 20 -	345.00 25.51 20 ‑4.48	337.08* 16.32 22 ‑6.67	324.16* 15.78 23 ‑10.25
	20 [a2]	Mean SD N %Diff	412.34 35.28 20 -	386.38 34.97 20 ‑6.30	373.00* 20.8 22 ‑9.54	358.10* 19.03 23 ‑13.15

[a] – Anova & Dunnett: * = p < 0.05

[a1] - Anova & Dunnett (Log): * = p < 0.05

[a2] - Anova & Dunnett (Rank): * = p < 0.05

Table 4.Summary of Maternal Body Weight Gain of Pregnant Rats

Sex: Female			G1 0 mg/kg/day	G2 125 mg/kg/day	G3 250 mg/kg/day	G4 500 mg/kg/day
Absolute Weight Gain (g)	0 → 3 [a]	Mean	19.09	17.88	17.72	19.58
		SD	5.40	5.76	3.91	4.08
		N	20	20	22	23
		%Diff	.	‑6.34	‑7.18	2.58
	3 → 5 [a1]	Mean	11.15	10.81	10.55	10.24
		SD	3.55	3.32	2.79	4.3
		N	20	20	22	23
		%Diff	.	‑3.02	‑5.39	‑8.16
	5 → 8 [a1]	Mean	10.88	4.58*	1.05*	‑10.54*
		SD	3.76	3.47	4.92	10.88
		N	20	20	22	23
		%Diff	.	‑57.91	‑90.37	‑196.89
	8 → 11 [a2]	Mean	18.7	17.1	14.92*	14.51*
		SD	5.22	3.23	5.76	5.2
		N	20	20	22	23
		%Diff	.	‑8.56	‑20.17	‑22.39
	11 → 14 [a2]	Mean	18.08	14.8	12.38*	13.73
		SD	5.53	7.19	6.62	6.36
		N	20	20	22	23
		%Diff	.	‑18.15	‑31.55	‑24.10
	14 → 17 [a1]	Mean	33	29.35	28.65	25.69
		SD	8.12	10.92	7.56	9.67
		N	20	20	22	23
		%Diff	.	‑11.05	‑13.18	‑22.14
	17 → 20 [a1]	Mean	51.17	41.37*	35.92*	33.94*
		SD	11.68	12.64	9.44	10.61
		N	20	20	22	23
		%Diff	.	‑19.14	‑29.80	‑33.67
Absolute Wei ght Gain (g)	0 → 5 [a]	Mean	30.23	28.69	28.26	29.82
		SD	7.18	5.36	4.04	5.49
		N	20	20	22	23
		%Diff	.	‑5.12	‑6.52	‑1.38
	5 → 20 [a1]	Mean	131.82	107.2*	92.91*	77.33*
		SD	23.96	28.6	20.32	18.95
		N	20	20	22	23
		%Diff	.	‑18.68	‑29.51	‑41.34
	0 → 20 [a]	Mean	162.05	135.89*	121.18*	107.14*
		SD	27.74	30.65	19.89	18.08
		N	20	20	22	23
		%Diff	.	‑16.15	‑25.22	‑33.88
Adjusted Bodyweight (g)	GD20 Bwt – gravid uterine weight[a2]	Mean	326.95	311.88*	304.61*	289.28*
		SD	24.88	15.26	17.17	13.06
		N	20	20	22	23
		%Diff	.	‑4.61	‑6.83	‑11.52
Adjusted Body weight Gain (g)	ADJ BWT‑GD 5 Body weight [a]	Mean	46.43	32.7*	24.53*	8.51*
		SD	11.64	11.08	13.02	10.76
		N	20	20	22	23
		%Diff	.	‑29.57	‑47.17	‑81.66

[a] – Anova & Dunnett: * = p < 0.05

[a1] - Anova & Dunnett (Rank): * = p < 0.05

[a2] - Anova & Dunnett (log): * = p < 0.05

Table 5.Summary of Food Consumption of Pregnant Rats

Sex: Female			G1 0 mg/kg/day	G2 125 mg/kg/day	G3 250 mg/kg/day	G4 500 mg/kg/day
Food Mean Consumption (g/day)	0 → 3 [a]	Mean	24.92	25.46	24.43	24.42
		SD	2.73	2.71	1.63	2.76
		N	20	20	22	23
		%Diff	.	2.15	‑1.98	‑2.01
	3 → 5 [a]	Mean	25.73	22.61*	18.7*	13.07*
		SD	2.64	2.41	4.66	4.94
		N	20	20	22	23
		%Diff	.	‑12.13	‑27.34	‑49.22
	5 → 8 [a1]	Mean	28.01	26.12	24.79*	20.57*
		SD	2.76	2.26	2.3	2.85
		N	20	20	22	23
		%Diff	.	‑6.76	‑11.51	‑26.57
	8 → 11 [a]	Mean	29.37	27.21*	26.26*	23.66*
		SD	2.89	2.07	1.92	2.87
		N	20	20	22	23
		%Diff	.	‑7.33	‑10.60	‑19.44
	11 → 14 [a]	Mean	31.65	29.68	28.69*	26.59*
		SD	2.77	3.36	2.44	2.76
		N	20	20	22	23
		%Diff	.	‑6.24	‑9.35	‑15.98
	14 → 17 [a]	Mean	33.02	29.69*	29.53*	27.16*
		SD	3.35	3.1	3.22	2.28
		N	20	20	22	23
		%Diff	.	‑10.07	‑10.57	‑17.76
	17 → 20 [a]	Mean	24.92	25.46	24.43	24.42
		SD	2.73	2.71	1.63	2.76
		N	20	20	22	23
		%Diff	.	2.15	‑1.98	‑2.01
Food Mean Consumption (g/day)	0 → 5 [a]	Mean	24.27	23.97	23.69	23.42
		SD	2.02	2.04	1.75	1.78
		N	20	20	22	23
		%Diff	.	‑1.26	‑2.38	‑3.54
	5 → 20 [a]	Mean	29.56	27.06*	25.59*	22.21*
		SD	2.68	2.06	1.9	1.95
		N	20	20	22	23
		%Diff	.	‑8.44	‑13.41	‑24.86
	0 → 20 [a]	Mean	28.24	26.29*	25.12*	22.51*
		SD	2.47	1.95	1.72	1.69
		N	20	20	22	23
		%Diff	.	‑6.90	‑11.04	‑20.28

[a] - Anova & Dunnett: * = p<0.05

[a1] - Anova & Dunnett (Rank): * = p<0.05

Table 6.Summary of Maternal Data

			G10 mg/kg/day	G2 125 mg/kg/day	G3 250 mg/kg/day	G4 500 mg/kg/day
Group size		N	24	24	24	24
Pregnant at C/S		N	20	20	22	23
Gravid Uterus Weight	[a]	Mean SD	85.391 21.348	74.498 24.843	68.386 * 23.504	68.814 * 15.232
Number of Corpora Lutea	[a1]	Mean SD Sum	17.4 2.2 348.0	16.8 3.1 335.0	16.8 1.8 370.0	17.1 1.9 394.0
Number of Implantation	[a]	Mean SD Sum	15.6 3.5 312.0	14.5 4.7 290.0	14.0 4.7 308.0	14.9 3.6 342.0
Number of dams with Early Resorption		N	9	10	12	13
Number of Early Resorptions	[a]	Mean SD Sum	0.8 1.1 15.0	1.0 1.3 19.0	0.8 0.9 17.0	0.9 1.0 21.0
% Early Resorption /Animal	[a]	Mean SD	4.64 6.62	7.53 10.39	5.65 8.38	6.74 8.44
Number of dams with Late Resorption		N	3	3	3	6
Number of Late Resorptions	[a]	Mean SD Sum	0.2 0.5 4.0	0.2 0.5 4.0	0.2 0.5 4.0	0.3 0.6 7.0
% Late Resorption /Animal	[a]	Mean SD	1.60 4.28	1.30 3.22	1.36 3.64	1.81 3.32
Number of dams with Resorptions	[f]	N	10	11	13	15
Total Number of Resorptions (Early + Late)	[f]	Mean SD Sum	1.0 1.3 19.0	1.2 1.7 23.0	1.0 1.1 21.0	1.2 1.3 28.0
Pre-implantation Loss/Animal	[a]	Mean SD	1.80 2.44	2.25 2.07	2.82 3.62	2.26 2.68
% Pre-implantation Loss	[a]	Mean SD	11.0 16.8	16.1 20.3	18.1 24.5	13.8 17.2
Post-implantation Loss/Animal	[a]	Mean SD	0.95 1.32	1.15 1.73	0.95 1.09	1.22 1.31
% Post-implantation Loss (%)	[a]	Mean SD	6.2 8.6	8.8 12.0	7.0 9.1	8.5 9.5

Note: Gross evaluation of placenta revealed no findings.

[a] - Anova & Dunnett (rank): * = p<0.05

[a1] Anova & Dunnett: * = p<0.05

[f]- Cochran Armitage, Chi-squared & Fisher's Exact

Table 7.Summary of Litter Data

Sex: Female			G10 mg/kg/day	G2 125 mg/kg/day	G3 250 mg/kg/day	G4 500 mg/kg/day
Total Number of fetuses		Sum	293	267	287	314
Total Number of Dead Fetuses		Sum	0	0	0	0
Total Number of Live fetuses		Sum	293	267	287	314
Live Male fetus		Sum	147	128	138	156
% Male Fetus			50.2	47.9	48.1	49.7
Mean Fetal Weight- Male (g)	[c]	Mean SD	3.755 0.304	3.551 0.328	3.350* 0.312	3.273* 0.545
Mean AGD- Male (mm)	[c]	Mean SD	2.55 0.10	2.57 0.12	2.49 0.12	2.43* 0.12
Live Female fetus		Sum	146	139	149	158
% Female Fetus			49.8	52.1	51.9	50.3
Mean Fetal Weight- Female (g)	[c1]	Mean SD	3.542 0.312	3.412 0.279	3.142* 0.306	3.079* 0.445
Mean AGD- Female (mm)	[c2]	Mean SD	0.94 0.06	0.93 0.06	0.91 0.06	0.89 0.08
Mean Fetal Weight -Male+Female (g)	[c3]	Mean SD	3.652 0.297	3.490 0.281	3.238* 0.300	3.172* 0.478
Mean AGD Male + Female (mm)	[c4]	Mean SD	1.73 0.23	1.75 0.26	1.66 0.19	1.64 0.18

[c] - Ancova/Anova & Dunnett(Rank): * = p < 0.05;{Covariate(s): Number of Live Male Fetuses}

[c1] - Ancova/Anova & Dunnett(Rank): * = p < 0.05;{Covariate(s): Number of Live Female Fetuses}

[c2] - Ancova/Anova & Dunnett;{Covariate(s): Number of Live Female Fetuses}

[c3] - Ancova/Anova & Dunnett(Rank): * = p < 0.05;{Covariate(s): Number of Live Fetuses}

[c4] - Ancova/Anova & Dunnett;Covariate(s): Number of Live Fetuses}

Table 8.Summary of Fetal External Observations

Exam Type: External     Number of Live Fetuses Examined: Number of Litters Evaluated:		G1 0 mg/kg/day	G2 125 mg/kg/day	G3 250 mg/kg/day	G4 500 mg/kg/day
		293 20	267 20	287 22	314 23
General Fetus, Small ‑ Anomaly	Fetuses N(%) Litters N(%)	1(0.3) 1(5.0)	0(0.0) 0(0.0)	0(0.0) 0(0.0)	0(0.0) 0(0.0)

[Fetuses N] ‑ Chi‑Squared & Chi‑Squared ‑ Pearson

[Litters N] ‑ Chi‑Squared & Chi‑Squared ‑ Pearson

Table 9.Summary of Fetal Visceral Observations

Exam Type: Fresh Visceral‑Body Only     Number of Live Fetuses Examined: Number of Litters Evaluated:		G1 0 mg/kg/day	G2 125 mg/kg/day	G3 250 mg/kg/day	G4 500 mg/kg/day
		142 20	127 20	138 22	150 23
Pelvis Ureter, Dilated, Moderate ‑ Anomaly   Abdomen Kidney, both, Renal pelvis dilation, Moderate ‑ Anomaly   Heart Heart, Innominate absent ‑ Malformation	Fetuses N(%) Litters N(%)   Fetuses N(%) Litters N(%)   Fetuses N(%) Litters N(%)	0(0.0) 0(0.0)   0(0.0) 0(0.0)   1(0.7) 1(5.0)	0(0.0) 0(0.0)   0(0.0) 0(0.0)   0(0.0) 0(0.0)	0(0.0) 0(0.0)   0(0.0) 0(0.0)   0(0.0) 0(0.0)	1(0.7) 1(4.3)   1(0.7) 1(4.3)   0(0.0) 0(0.0)

[Fetuses N] ‑ Chi‑Squared & Chi‑Squared ‑ Pearson

[Litters N] ‑ Chi‑Squared & Chi‑Squared ‑ Pearson

Table 10.Summary of Fetal Skeletal Observations

Exam Type: Skeletal‑Entire     Number of Live Fetuses Examined: Number of Litters Evaluated:		G1 0 mg/kg/day	G2 125 mg/kg/day	G3 250 mg/kg/day	G4 500 mg/kg/day
		151 20	137 20	149 22	164 23
Pelvic girdle Pubis, both, Incomplete ossification - Variation   Ischium, Delayed skeletal ossification - Variation   Caudal vertebrae 4th caudal centrum, Delayed skeletal ossification - Variation   3rd caudal centrum, Delayed skeletal ossification ‑ Variation   2nd caudal centrum, Delayed skeletal ossification ‑ Variation   1st caudal centrum, Delayed skeletal ossification ‑ Variation   2nd caudal arch, Delayed skeletal ossification ‑ Variation   1st caudal arch, Delayed skeletal ossification ‑ Variation   sacral vertebrae 4th sacral centrum, Delayed skeletal ossification ‑ Variation   3rd sacral centrum, Delayed skeletal ossification ‑ Variation   4th sacral arch, Delayed skeletal ossification ‑ Variation   3rd sacral arch, Delayed skeletal ossification ‑ Variation   Lumbar vertebrae 2nd lumbar centrum, Dumbbell‑shaped ‑ Anomaly   1st lumbar centrum, Dumbbell‑shaped ‑ Anomaly   7th lumbar centrum and arch, Extra ‑ Anomaly   thoracic vertebrae 13th thoracic centrum, Split – Anomaly   13th thoracic centrum, Dumbbell‑shaped ‑ Anomaly   12th thoracic centrum, Split ‑ Anomaly   12th thoracic centrum, Dumbbell‑shaped ‑ Anomaly   11th thoracic centrum, Split ‑ Anomaly   11th thoracic centrum, Dumbbell‑shaped ‑ Anomaly   11th thoracic centrum, Delayed skeletal ossification ‑ Variation   10th thoracic centrum, Split ‑ Anomaly   10th thoracic centrum, Dumbbell‑shaped ‑ Anomaly   10th thoracic centrum, Delayed skeletal ossification ‑ Variation   9th thoracic centrum, Split ‑ Anomaly   9th thoracic centrum, Dumbbell‑shaped ‑ Anomaly   9th thoracic centrum, Delayed skeletal ossification ‑ Variation   8th thoracic centrum, Split ‑ Anomaly   8th thoracic centrum, Dumbbell‑shaped ‑ Anomaly   8th thoracic centrum, Delayed skeletal ossification ‑ Variation   7th thoracic centrum, Dumbbell‑shaped ‑ Anomaly   7th thoracic centrum, Delayed skeletal ossification ‑ Variation   6th thoracic centrum, Split ‑ Anomaly   6th thoracic centrum, Dumbbell‑shaped ‑ Anomaly   6th thoracic centrum, Delayed skeletal ossification ‑ Variation   5th thoracic centrum, Dumbbell‑shaped – Anomaly   5th thoracic centrum, Delayed skeletal ossification ‑ Variation   4th thoracic centrum, Split ‑ Anomaly   4th thoracic centrum, Dumbbell‑shaped ‑ Anomaly   4th thoracic centrum, Delayed skeletal ossification ‑ Variation   3rd thoracic centrum, Dumbbell‑shaped ‑ Anomaly   3rd thoracic centrum, Delayed skeletal ossification ‑ Variation   3rd thoracic centrum, Incomplete ossification ‑ Variation   2nd thoracic centrum, Delayed skeletal ossification ‑ Variation   1st thoracic centrum, Delayed skeletal ossification ‑ Variation   1st thoracic centrum, Incomplete ossification ‑ Variation   Ribs 14th rib, Right, Rudimentary - Anomaly   14th rib, Right, Accessory - Anomaly   14th rib, Left, Rudimentary - Anomaly   14th rib, Left, Accessory - Anomaly   14th rib, Left, Extra - Anomaly   14th rib, Bilateral, Rudimentary - Anomaly   14th rib, Bilateral, Accessory - Anomaly   14th rib, Bilateral, Extra ‑ Anomaly   13th rib, Bilateral, Wavy Rib, Moderate ‑ Anomaly   12th rib, Bilateral, Wavy Rib, Moderate - Anomaly   11th rib, Bilateral, Wavy Rib, Moderate ‑ Anomaly   10th rib, Bilateral, Wavy Rib, Moderate - Anomaly   9th rib, Right, Wavy Rib, Slight - Anomaly   9th rib, Bilateral, Wavy Rib, Moderate ‑ Anomaly   8th rib, Right, Wavy Rib, Slight ‑ Anomaly   8th rib, Bilateral, Wavy Rib, Moderate ‑ Anomaly   7th rib, Bilateral, Wavy Rib, Slight ‑ Anomaly   7th rib, Bilateral, Wavy Rib, Moderate ‑ Anomaly   6th rib, Bilateral, Wavy Rib, Slight ‑ Anomaly   6th rib, Bilateral, Wavy Rib, Moderate ‑ Anomaly   5th rib, Bilateral, Wavy Rib, Slight ‑ Anomaly   5th rib, Bilateral, Wavy Rib, Moderate ‑ Anomaly   4th rib, Bilateral, Wavy Rib, Moderate ‑ Anomaly   Sternebrae 6th sternebra, Delayed skeletal ossification ‑ Variation   6th sternebra, Incomplete ossification ‑ Variation   5th sternebra, Hypoplastic ‑ Anomaly   5th sternebra, Asymmetrical ossification ‑ Anomaly   5th sternebra, Split ‑ Anomaly   4th sternebra, Hypoplastic ‑ Anomaly   4th sternebra, Delayed skeletal ossification ‑ Variation   4th sternebra, Asymmetrical ossification ‑ Anomaly   4th sternebra, Incomplete ossification ‑ Variation   4th sternebra, Split incomplete ossification ‑ Anomaly   3rd sternebra, Delayed skeletal ossification ‑ Variation   3rd sternebra, Asymmetrical ossification ‑ Anomaly   3rd sternebra, Incomplete ossification ‑ Variation   3rd sternebra, Split incomplete ossification ‑ Anomaly   2nd sternebra, Hypoplastic ‑ Anomaly   2nd sternebra, Asymmetrical ossification ‑ Anomaly   2nd sternebra, Split ‑ Anomaly   2nd sternebra, Split incomplete ossification ‑ Anomaly   1st sternebra, Delayed skeletal ossification ‑ Variation   1st sternebra, Incomplete ossification ‑ Variation   Skull Supraoccipital, Incomplete ossification ‑ Variation   Squamosal, Incomplete ossification ‑ Variation   Parietal, Incomplete ossification ‑ Variation   Interparietal, Incomplete ossification ‑ Variation   Hyoid, Incomplete ossification ‑ Variation   Frontal, Incomplete ossification ‑ Variation	Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%)   Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%)   Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%)   Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%)   Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%)   Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%)   Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%)   Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%) Fetuses N(%) Litters N(%)	1(0.7) 1(5.0) 1(0.7) 1(5.0)   1(0.7) 1(5.0) 1(0.7) 1(5.0) 1(0.7) 1(5.0) 1(0.7) 1(5.0) 1(0.7) 1(5.0) 1(0.7) 1(5.0)   1(0.7) 1(5.0) 1(0.7) 1(5.0) 1(0.7) 1(5.0) 1(0.7) 1(5.0)   0(0.0) 0(0.0) 2(1.3) 2(10.0) 0(0.0) 0(0.0)   0(0.0) 0(0.0) 5(3.3) 4(20.0) 3(2.0) 3(15.0) 17(11.3) 11(55.0) 4(2.6) 3(15.0) 19(12.6) 13(65.0) 0(0.0) 0(0.0) 1(0.7) 1(5.0) 15(9.9) 12(60.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 10(6.6) 7(35.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 5(3.3) 4(20.0) 0(0.0) 0(0.0) 1(0.7) 1(5.0) 0(0.0) 0(0.0) 1(0.7) 1(5.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 3(2.0) 3(15.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 1(0.7) 1(5.0) 0(0.0) 0(0.0) 1(0.7) 1(5.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 1(0.7) 1(5.0)   10(6.6) 7(35.0) 0(0.0) 0(0.0) 11(7.3) 8(40.0) 0(0.0) 0(0.0) 2(1.3) 2(10.0) 15(9.9) 8(40.0) 0(0.0) 0(0.0) 6(4.0) 3(15.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)   3(2.0) 3(15.0) 59(39.1) 18(90.0) 16(10.6) 11(55.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 1(0.7) 1(5.0) 1(0.7) 1(5.0) 4(2.6) 4(20.0) 0(0.0) 0(0.0) 1(0.7) 1(5.0) 1(0.7) 1(5.0) 1(0.7) 1(5.0) 0(0.0) 0(0.0) 2(1.3) 2(10.0) 1(0.7) 1(5.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 1(0.7) 1(5.0) 1(0.7) 1(5.0)   6(4.0) 3(15.0) 1(0.7) 1(5.0) 12(7.9) 6(30.0) 11(7.3) 5(25.0) 9(6.0) 8(40.0) 0(0.0) 0(0.0)	0(0.0) 0(0.0) 0(0.0) 0(0.0)   0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)   0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)   0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)   1(0.7) 1(5.0) 1(0.7) 1(5.0) 2(1.5) 2(10.0) 7(5.1) 6(30.0) 5(3.6) 3(15.0) 16(11.7) 11(55.0) 0(0.0) 0(0.0) 1(0.7) 1(5.0) 25(18.2) 15(75.0) 0(0.0) 0(0.0) 1(0.7) 1(5.0) 10(7.3) 7(35.0) 0(0.0) 0(0.0) 1(0.7) 1(5.0) 5(3.6) 5(25.0) 0(0.0) 0(0.0) 1(0.7) 1(5.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 1(0.7) 1(5.0) 1(0.7) 1(5.0)   5(3.6) 5(25.0) 0(0.0) 0(0.0) 9(6.6) 8(40.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 12(8.8) 8(40.0) 0(0.0) 0(0.0) 2(1.5) 2(10.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)   7(5.1) 4(20.0) 62(45.3) 18(90.0) 20(14.6) 10(50.0) 1(0.7) 1(5.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 7(5.1) 5(25.0) 6(4.4) 2(10.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 7(5.1) 6(30.0) 6(4.4)* 2(10.0) 0(0.0) 0(0.0) 9(6.6)* 8(40.0)* 1(0.7) 1(5.0) 1(0.7) 1(5.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 2(1.5) 1(5.0)   8(5.8) 7(35.0) 1(0.7) 1(5.0) 19(13.9) 10(50.0) 14(10.2) 9(45.0) 10(7.3) 8(40.0) 1(0.7) 1(5.0)	0(0.0) 0(0.0) 0(0.0) 0(0.0)   0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)   0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)   1(0.7) 1(4.5) 0(0.0) 0(0.0) 0(0.0) 0(0.0)   1(0.7) 1(4.5) 4(2.7) 4(18.2) 0(0.0) 0(0.0) 11(7.4) 10(45.5) 2(1.3) 2(9.1) 23(15.4) 13(59.1) 2(1.3) 2(9.1) 1(0.7) 1(4.5) 19(12.8) 15(68.2) 1(0.7) 1(4.5) 0(0.0) 0(0.0) 10(6.7) 8(36.4) 1(0.7) 1(4.5) 0(0.0) 0(0.0) 5(3.4) 5(22.7) 1(0.7) 1(4.5) 2(1.3) 2(9.1) 1(0.7) 1(4.5) 0(0.0) 0(0.0) 1(0.7) 1(4.5) 1(0.7) 1(4.5) 0(0.0) 0(0.0) 1(0.7) 1(4.5) 1(0.7) 1(4.5) 0(0.0) 0(0.0) 1(0.7) 1(4.5) 0(0.0) 0(0.0) 1(0.7) 1(4.5) 0(0.0) 0(0.0) 2(1.3) 2(9.1) 7(4.7)* 4(18.2) 2(1.3) 2(9.1)   6(4.0) 6(27.3) 1(0.7) 1(4.5) 7(4.7) 6(27.3) 3(2.0) 3(13.6) 0(0.0) 0(0.0) 15(10.1) 8(36.4) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 2(1.3) 2(9.1) 2(1.3) 2(9.1) 2(1.3) 2(9.1) 2(1.3) 2(9.1) 1(0.7) 1(4.5) 2(1.3) 2(9.1) 1(0.7) 1(4.5) 2(1.3) 2(9.1) 1(0.7) 1(4.5) 2(1.3) 2(9.1) 1(0.7) 1(4.5) 2(1.3) 2(9.1) 1(0.7) 1(4.5) 2(1.3) 2(9.1) 2(1.3) 2(9.1)   7(4.7) 5(22.7) 92(61.7)* 20(90.9) 16(10.7) 9(40.9) 0(0.0) 0(0.0) 2(1.3) 2(9.1) 0(0.0) 0(0.0) 1(0.7) 1(4.5) 3(2.0) 3(13.6) 12(8.1)* 8(36.4) 0(0.0) 0(0.0) 1(0.7) 1(4.5) 3(2.0) 3(13.6) 8(5.4)* 5(22.7) 0(0.0) 0(0.0) 14(9.4)* 8(36.4)* 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 1(0.7) 1(4.5)   23(15.4)* 10(45.5) 1(0.7) 1(4.5) 29(19.5)* 14(63.6) 34(22.8)* 14(63.6) 23(15.4)* 12(54.5) 0(0.0) 0(0.0)	0(0.0) 0(0.0) 0(0.0) 0(0.0)   0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)   0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)   0(0.0) 0(0.0) 1(0.6) 1(4.3) 2(1.2) 1(4.3)   1(0.6) 1(4.3) 7(4.3) 4(17.4) 1(0.6) 1(4.3) 14(8.5) 10(43.5) 1(0.6) 1(4.3) 31(18.9) 18(78.3) 0(0.0) 0(0.0) 2(1.2) 2(8.7) 27(16.5) 14(60.9) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 29(17.7)* 11(47.8) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 8(4.9) 6(26.1) 0(0.0) 0(0.0) 6(3.7) 5(21.7) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 2(1.2) 2(8.7) 0(0.0) 0(0.0) 2(1.2) 2(8.7) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 1(0.6) 1(4.3) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 1(0.6) 1(4.3) 0(0.0) 0(0.0) 4(2.4) 3(13.0) 5(3.0) 4(17.4)   12(7.3) 6(26.1) 2(1.2) 2(8.7) 6(3.7) 6(26.1) 5(3.0) 2(8.7) 0(0.0) 0(0.0) 20(12.2) 9(39.1) 4(2.4) 3(13.0) 5(3.0) 4(17.4) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)   20(12.2)* 7(30.4) 80(48.8) 20(87.0) 17(10.4) 11(47.8) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 1(0.6) 1(4.3) 1(0.6) 1(4.3) 4(2.4) 3(13.0) 38(23.2)* 14(60.9)* 2(1.2) 2(8.7) 0(0.0) 0(0.0) 4(2.4) 3(13.0) 36(22.0)* 11(47.8)* 1(0.6) 1(4.3) 21(12.8)* 14(60.9)* 1(0.6) 1(4.3) 0(0.0) 0(0.0) 1(0.6) 1(4.3) 0(0.0) 0(0.0) 19(11.6)* 5(21.7)   18(11.0)* 9(39.1) 4(2.4) 4(17.4) 21(12.8) 10(43.5) 23(14.0) 10(43.5) 5(3.0) 4(17.4) 3(1.8) 2(8.7)

[Fetuses N] ‑ Chi‑Squared & Chi‑Squared ‑ Pearson: * = p<0.05

[Litters N] ‑ Chi‑Squared & Chi‑Squared ‑ Pearson: * = p<0.05

Conclusions:

In this OECD 414 study conducted with Wistar rats, the NOAELs for maternal developmental toxicity and foetal developmental toxicity were considered at 500 and 125 mg/kg/day, respectively. NOAEL for maternal general toxicity could not be established due to significant decreases in body weight gain and food intake at 125 mg/kg/day. The fetal effects observed at 250 and 500 mg/kg/day were limited to decreased fetal weights (by about 11% at 250 mg/kg/day and by about 13% at 500 mg/kg/day). These fetal effects were considered secondary to maternal toxicity, which at 250 and 500 mg/kg/day included significant decreases in body weight gain during different periods of gestation e.g. GD 5-20 (by 19% at 250 mg/kg, and by 29% at 500 mg/kg), significant decreases in absolute body weight (by 5-10% at 250 mg/kg/day, and by 7-13% at 500 mg/kg/day), and significant reductions in food intake during different periods of gestation (by 9-27% at 250 mg/kg/day, and by 16-49% at 500 mg/kg).

Executive summary:

In this study performed according to OECD 414 (2018) and GLP, the test material, containing 99.74% of N-Tert-Butylacrylamide (CAS No. 107-58-4), was given by oral gavage to 24 mated female Wistar rats per dose at 0 (vehicle control; milli-Q water), 125, 250 and 500 mg/kg bw/day from GD 5 to 19. The following parameters and endpoints were evaluated: mortality, clinical signs, body weights, body weight gains, food consumption, gross pathology, gravid uterine weights, intrauterine growth and survival, number of corpora lutea, and fetal parameters [sex, weight and anogenital distance, and external, visceral, and skeletal observations]. Approximately half the number of the fetuses from each litter were examined for visceral malformations and variations and the remaining half were evaluated for skeletal malformations and variations. In addition, from each dam, the thyroid was weighed and subjected to microscopic evaluation. Thyroid hormones levels were determined from blood samples collected at terminal sacrifice (on GD 20).

Main findings from the study are summarized below:

Mortality, clinical signs and gross necropsy changes: There were no unscheduled deaths, clinical signs, or gross necropsy findings in any of the dams.

Body weight: Significant decreases in maternal body weight were observed at 250 mg/kg (On GD 11, 14, 17 and 20) and 500 mg/kg (On GD 8, 11, 14, 17 and 20). The decreases at 250 mg/kg were in the range of 5 to 10% whereas the decreases at 500 mg/kg were in the range of 7 to 13%. No significant decrease in maternal body weight were observed at 125 mg/kg. In terms of maternal body weight gain, significant decreases were observed from GD 5 to 20 at 125 mg/kg (by 18.7%), 250 mg/kg (by 29.5%) and at 500 mg/kg (by 41.3%). Food consumption: As compared to the vehicle control group, significant decreases in mean food consumption (in the range of 6 to 49%) were observed in all treated groups during different periods of gestation.

Maternal Parameters: No significant changes in gravid uterine weight, number of corpora lutea, implantations, early and late resorptions, pre- and post-implantation loss were observed at 125 mg/kg. Significant decreases in uterine weight were observed at 250 mg/kg (by 20%) and 500 mg/kg (by 19%) compared to the control. No other significant changes in maternal developmental were observed at 250 or 500 mg/kg. No gross pathological effects on the placenta were observed at any dose level.

Litter Parameters: No significant changes in total number of fetuses, total number of live fetuses, total number of dead fetuses, sex ratio, AGD, or fetal weight were observed at 125 mg/kg. Significant decreases in male fetal weight, female fetal weight, and sex-combined fetal weight were observed at 250 mg/kg (by approx. 11% for all cases) and 500 mg/kg (by approx. 13% for all cases). A small, but statistically significant decrease in male AGD (by less than 5%) was observed at 500 mg/kg. No other significant changes in litter data were observed at 250 or 500 mg/kg.

Fetal examination: The results from the external, visceral, and skeletal examinations revealed no significant effects that could be attributed to the test item.

No significant changes in thyroid hormone levels (T3, T4 and TSH), thyroid weights, or thyroid histology were observed at any dose level. Based on the above findings, under the test conditions and doses employed in this study, the following NOAEL values were concluded:

NOAEL for maternal systemic toxicity could not be established due to significant decreases in maternal body weight gain and maternal food intake at 125 mg/kg/day and above. Since no significant changes were observed in mean maternal body weight at 125 mg/kg/day, the changes in body weight gain and food intake at 125 mg/kg/day were likely just slightly above the threshold level of toxicological concern.

NOAEL for maternal developmental toxicity was considered at 500 mg/kg/day since no significant effects were observed on the averages for abortions, pre-implantation loss, post-implantation loss, early resorptions, late resorptions, total resorptions, corpora lutea, number of dead foetuses, or gross pathology.

NOAEL for fetal developmental toxicity was 125 mg/kg/day due to significant decreases fetal weight at ≥250 mg/kg/day.

NOAEL for teratogenicity was 500 mg/kg/day as the results from the fetal external, visceral, and skeletal examinations did not reveal any adverse effects of treatment.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

125 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1

Additional information

Study 1

In this study performed according to OECD 414 (2018) and GLP, the test material, containing 99.74% of N-Tert-Butylacrylamide (CAS No. 107-58-4), was given by oral gavage to 24 mated female Wistar rats per dose at 0 (vehicle control; milli-Q water), 125, 250 and 500 mg/kg bw/day from GD 5 to 19. The following parameters and endpoints were evaluated: mortality, clinical signs, body weights, body weight gains, food consumption, gross pathology, gravid uterine weights, intrauterine growth and survival, number of corpora lutea, and fetal parameters [sex, weight and anogenital distance, and external, visceral, and skeletal observations]. Approximately half the number of the fetuses from each litter were examined for visceral malformations and variations and the remaining half were evaluated for skeletal malformations and variations. In addition, from each dam, the thyroid was weighed and subjected to microscopic evaluation. Thyroid hormones levels were determined from blood samples collected at terminal sacrifice (on GD 20).

Main findings from the study are summarized below:

Mortality, clinical signs and gross necropsy changes: There were no unscheduled deaths, clinical signs, or gross necropsy findings in any of the dams.

Body weight: Significant decreases in maternal body weight were observed at 250 mg/kg (On GD 11, 14, 17 and 20) and 500 mg/kg (On GD 8, 11, 14, 17 and 20). The decreases at 250 mg/kg were in the range of 5 to 10% whereas the decreases at 500 mg/kg were in the range of 7 to 13%. No significant decrease in maternal body weight were observed at 125 mg/kg. In terms of maternal body weight gain, significant decreases were observed from GD 5 to 20 at 125 mg/kg (by 18.7%), 250 mg/kg (by 29.5%) and at 500 mg/kg (by 41.3%). Food consumption: As compared to the vehicle control group, significant decreases in mean food consumption (in the range of 6 to 49%) were observed in all treated groups during different periods of gestation.

Maternal Parameters: No significant changes in gravid uterine weight, number of corpora lutea, implantations, early and late resorptions, pre- and post-implantation loss were observed at 125 mg/kg. Significant decreases in uterine weight were observed at 250 mg/kg (by 20%) and 500 mg/kg (by 19%) compared to the control. No other significant changes in maternal developmental were observed at 250 or 500 mg/kg. No gross pathological effects on the placenta were observed at any dose level.

Litter Parameters: No significant changes in total number of fetuses, total number of live fetuses, total number of dead fetuses, sex ratio, AGD, or fetal weight were observed at 125 mg/kg. Significant decreases in male fetal weight, female fetal weight, and sex-combined fetal weight were observed at 250 mg/kg (by approx. 11% for all cases) and 500 mg/kg (by approx. 13% for all cases). A small, but statistically significant decrease in male AGD (by less than 5%) was observed at 500 mg/kg. No other significant changes in litter data were observed at 250 or 500 mg/kg.

Fetal examination: The results from the external, visceral, and skeletal examinations revealed no significant effects that could be attributed to the test item.

No significant changes in thyroid hormone levels (T3, T4 and TSH), thyroid weights, or thyroid histology were observed at any dose level. Based on the above findings, under the test conditions and doses employed in this study, the following NOAEL values were concluded:

NOAEL for maternal systemic toxicity could not be established due to significant decreases in maternal body weight gain and maternal food intake at 125 mg/kg/day and above. Since no significant changes were observed in mean maternal body weight at 125 mg/kg/day, the changes in body weight gain and food intake at 125 mg/kg/day were likely just slightly above the threshold level of toxicological concern.

NOAEL for maternal developmental toxicity was considered at 500 mg/kg/day since no significant effects were observed on the averages for abortions, pre-implantation loss, post-implantation loss, early resorptions, late resorptions, total resorptions, corpora lutea, number of dead foetuses, or gross pathology.

NOAEL for fetal developmental toxicity was 125 mg/kg/day due to significant decreases fetal weight at ≥250 mg/kg/day.

NOAEL for teratogenicity was 500 mg/kg/day as the results from the fetal external, visceral, and skeletal examinations did not reveal any adverse effects of treatment.

Justification for classification or non-classification

In the OECD 414 study, adverse effects on development were limited to decreased foetal weights in males and females, respectively, and in total (i.e. male and females combined) at 250 mg/kg (by approx. 11% for all cases vs. control data) and 500 mg/kg (by approx. 13% for all cases vs. control data). These effects occurred in the presence of maternal systemic toxicity, which included significant decreases in absolute body weight at 250 mg/kg (by 5-10% vs. control data on GDs 11, 14, 17 and 20) and 500 mg/kg (by 7-13% vs. control data on GDs 8, 11, 14, 17 and 29); significant decreases in body weight gain during several periods of gestation, e.g. from GD 5-20 at 250 mg/kg (by 19% vs. control data) and from GD 5-20 at 500 mg/kg (by 29% vs. control data); and significant reductions in food intake during different periods of gestation at 250 mg/kg (by 9 to 27% vs. control data) and 500 mg/kg (by 16 to 49% vs. control data).

In the literature, there is a well-established relationship between lower foetal weights and reduced maternal weight gains late in gestation (Chernoff et al. Reproductive Toxicology 2008; 25: 192-202). In the presented OECD 414 study, decreased maternal body weight gains were especially pronounced during the GD 17-20 period at 250 mg/kg (mean gain, 35.9 gram [i.e. a 30% decrease vs. control data]) and 500 mg/kg (mean gain, 33.9 gram [i.e. a 33.8% decrease]) compared to the control group (mean gain, 51.2 gram). On this basis we consider the lower foetal weights observed at 250 mg/kg (by approx. 11% vs control data) and 500 mg/kg (by approx. 13% vs control data) to be secondary to maternal general toxicity and not relevant for classification.

In the OECD 414 study, decreased gravid uteri weights were observed at 250 mg/kg (mean, 68.386 gram) and 500 mg/kg (mean, 68.814 gram) compared to the control (mean 85.391 gram). This effect was not associated with any gross lesions and could not be correlated with any developmental effects except for lower foetal weights that we attributed to lower maternal body weight gains during late gestation. The effect on uterus weight at 250 and 500 mg/kg was therefore not considered to be of any biological significance in the study.

In the OECD 407 study, no adverse effects on reproductive organ tissue were observed. There was a trend of lower uterus weight in the main study (i.e. mean uteri weights of 0.720, 0.645, 0.561, and 0.553 gram at 0, 125, 250 and 500 mg/kg, respectively) but not in the recovery groups (i.e. mean uteri weights of 0.777 and 0.723 gram at 0 and 500 mg/kg, respectively).

Based on the presented data, the substance is regarded to be classified as Not Classified for Toxicity to reproduction according to Regulation EC No 1272/2008.