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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Only draft final study report available at submission.
Reliability:
1 (reliable without restriction)
Justification for type of information:
Following ECHA 's communication CCH-D-2114541355-52-01/F:
A PNDT study according to the test method OECD TG 414 must be performed in rabbit as preferred non-rodent species. The study shall be performed with oral administration of the analogue substance ammonium sulfanylacetate (EC 226-540-9).
The study has been conducted on sodium sulfanylacetate (EC 206-696-4), by the registrants of the substance (Bruno Bock Chemische Fabrik GmbH & Co. KG).
At the moment only the draft study report is available.


HYPOTHESIS FOR THE ANALOGUE APPROACH

- The target and source substances are grouped based on similar molecular structure and functionality.

- The target and source substance have similar physico-chemical properties.

- The target and source substances have similar reactivity :
o At acidic pH (for example the pH stomach of 1.2), more than 99% of the product will be present as an acidic form.
o At neutral pH, diluted solutions of thioglycolic acid and its salts undergo full dissociation into the thioglycolate anion (HS-CH2-COO-) and the respective cations (H+, NH4+ or Na+).

- The target and source substances have similar toxicity :
o The toxicity of each compound is driven by the thioglycolate anion.
o The effect of the counter-ion (sodium or ammonium) on the systemic toxicity of the thioglycolic salts is not expected to be significant.
o The acute and repeated dose toxicity of sodium sulphate and ammonium chloride, sulphate and phosphate is moderate, with effective dose levels far higher than those of the thioglycolates.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

1
Chemical structure
Reference substance name:
Sodium mercaptoacetate
EC Number:
206-696-4
EC Name:
Sodium mercaptoacetate
Cas Number:
367-51-1
Molecular formula:
C2H4O2S.Na
IUPAC Name:
sodium sulfanylacetate
Test material form:
liquid

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: KCC BIO LABS, Sy No.8/1, Tondagere Village, Tavarakere Post, Hebburu Hobli Tumakuru- 572120 Karnataka, India

- Age at study initiation: 8 - 9 months

- Weight at study initiation:
Mean body weight Body weight range
G1 – 3.7606 ± 0.171 3.4146 to 4.0913
G2 – 3.7541 ± 0.166 3.4738 to 4.0832
G3 – 3.7533 ± 0.160 3.4347 to 4.9955
G4 – 3.7577 ± 0.178 3.3405 to 4.0942

- Fasting period before study: none

- Housing:
Standard laboratory conditions, air conditioned with adequate fresh air supply (12 - 15 air changes/hour). Environment: with temperature 19 to 21°C, relative humidity 64 to 65 %, with 12 hours light and 12 hours dark cycle.
The maximum and minimum temperature and relative humidity in the experimental rooms were (females and males were housed in different rooms) recorded once daily. The relative humidity in the experimental rooms was calculated daily from dry and wet bulb temperature recordings.

- Diet (e.g. ad libitum):
Rabbit feed manufactured by Special Diets Services, P.O Box 705, Witham, Essex, CM8 3AD, England (Batch No.5286) was provided ad libitum in stainless steel feed hoppers to rabbits.

- Water (e.g. ad libitum):
Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard water filter-cum-purifier (Eureka Forbes Ltd, Mumbai, India) was provided ad libitum in polycarbonate bottles with stainless steel sipper tubes to rabbits.

- Acclimation period:
After clinical examination for good health and suitability for the study, the rabbits were acclimatized for five days before initiation of mating. During the acclimatization period, all rabbits were observed at least once daily. Females used in this study were nulliparous and non-pregnant.


IN-LIFE DATES:
Acclimatization : Start: 05 May 2022 / End: 09 May 2022
Co-habitation : Start: 10 May 2022 / End: 19 May 2022
Treatment : Start: 16 May 2022 / End: 26 June 2022
Sacrifice : Start: 08 June 2022 / End: 17 June 2022

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Milli-Q water. The concentration and stability of the test item in the vehicle (Milli-Q water) was performed at dose concentrations of 1.0 0 mg/mL and 150 mg/mL. The test item was found to be stable in the vehicle up to 24 h.
Details on exposure:
VEHICLE
- Concentration in vehicle: 2.5 / 7.5 / 15 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item dose formulations were prepared daily prior to treatment and used within the stability period.
For homogeneity and concentration analysis of the test item, the prepared formulations were sampled at the initiation (16 May 2022) of treatment (GD 6) and at termination (14 June 2022) of treatment period (GD 28). Prepared formulations were samples in duplicate sets (one set for analysis and one set as back-up). Dose formulation samples were analysed for Thiocare®H102 46% formulation content using analytical validated method.
Dose formulations were considered acceptable as the mean percent recovery results were within 70 to 120% at each dose level and relative standard deviation (% RSD), of assay was less than or equal to 20%.
Details on mating procedure:
During the mating period, females were cohabited randomly with males in a 1:1 ratio. After confirmation of mating visually, the Gestation Day 0 (GD 0) was recorded for each individual rabbit.
Duration of treatment / exposure:
From GD 6 to GD 28 of presumed gestation at approximately the same time each day (varying by ± 3 hours).
Frequency of treatment:
Daily
Duration of test:
GD 0 to GD 29
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control group
Dose / conc.:
5 mg/kg bw/day
Remarks:
4 mg/kg/day as mercaptoacetic acid
Dose / conc.:
15 mg/kg bw/day
Remarks:
12 mg/kg/day as mercaptoacetic acid
Dose / conc.:
30 mg/kg bw/day
Remarks:
24 mg/kg/day as mercaptoacetic acid
No. of animals per sex per dose:
23 rabbits/group
Control animals:
yes, concurrent vehicle
Details on study design:
A preliminary dose-range-finding (DRF) study in pregnant rabbits was carried out using 6 rabbits per group . The rabbits were treated at doses of 25 (G2), 50 (G3), 75(G4) and 100 (G5) mg/kg/day along with vehicle control (Milli-Q water) group at a dose volume of 2 mL/kg body weight.

The results were as follows:

Oral administration of Thiocare® H102 46% at =50 mg/kg/day to pregnant rabbits resulted in unscheduled mortalities which required humane removal of rabbits at =75 mg/kg/day. At 25 mg/kg/day, there were no clinical signs or mortalities. The mean body weight and body weight gains were lower with significant reduction in the food consumption, indicative of maternal toxicity. Maternal parameters (uterine weight, number of corpora lutea, implantations, early and late resorptions, and pre- and post-implantation loss) and litter parameters (mean fetal weights) were comparable to control. There was one doe at 25 mg/kg/day which had complete resorption. There were no external fetal malformations observed at 25 and 50 mg/kg/day doses. The gross pathology findings in stomach (red foci in glandular mucosa) and kidneys (red foci) at =50 mg/kg/day were related to test item administration.

Based on these results of dose range finding study in pregnant rabbits and considering treatment related deaths at =50 mg/kg/day, following doses are proposed for the definitive study in pregnant rabbits in consultation with sponsor:

G1 - Vehicle control - 0 mg/kg/day
G2 - Low dose - 5 mg/kg/day
G3 - Mid dose - 15 mg/kg/day
G4 - High dose - 30 mg/kg/day

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The rabbits were observed for morbidity and mortality twice daily, i.e., once in the morning and once in the afternoon.
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: during the treatment period of the study: pre-dose and post dose (within 1-2 hours of administration).

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes for the following intervals: GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29


WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: Gross pathological changes of all external and visceral organs of rabbits, including uterine contents were recorded

OTHER:
Aborted rabbit was necropsied, and gross observations observed were recorded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: gross evaluation of placenta, pregnancy status

Uteri that appeared non-gravid were subjected to 10% ammonium sulfide staining to observe implantation sites (identified as pregnant animals) or to confirm the non-pregnant status
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all live fetuses]
- Head examinations: Yes: [half per litter ]
- Anogenital distance of all live rodent pups: No
Statistics:
The numerical results were evaluated by an appropriate statistical method using the litter as the unit for data analysis.

The following statistical tests were used:
Data was captured using the ProvantisTM laboratory information management system (LIMS), parameters such as maternal body weight, body weight change, food consumption, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, Pre/post implantation loss, number of implantations, sex ratio, number of corpora lutea, early and late resorptions was evaluated using the Levene Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. Data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA), if data found to be nonhomogeneous or of nonnormal data was subjected for transformation and ANOVA was done on transformed data. When ANOVA was significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.
Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group.

The incidence of does with resorptions was 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 was tested for Cochran Armitage trend test and pair wise comparison was be done by Fisher’s exact test.

Descriptive statistics Mean, SD, Percentages & Numbers was be presented by Treatment group and Day.

All hypothesis testing wase carried out at the 5% (two-sided) significance level unless otherwise specified. Significant differences are designated throughout the report as below:

*: Statistically significant difference from the control group at p < 0.05

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no clinical signs or mortalities at 5 and 15 mg/kg/day. At 30 mg/kg/day, hypoactivity was observed on two rabbits one rabbit RBa8653 was found hypoactive on GD 25 and aborted on GD 28, and another rabbit RBa8654 was hypoactive from GD 15-20 and 25 to 27 and posture sitting with head hung down from GD 15 to 18.
Mortality:
no mortality observed
Description (incidence):
The number of rabbits sacrificed at term were 21, 22, 22 and 20 (G1 to G4), of which 1, 3, 2 and 1 rabbits were non-pregnant and the number of pregnant rabbits were 20, 19, 20 and 19, at 0, 5, 15 and 30 mg/kg/day, respectively.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean maternal body weights and body weight gains during the different days of gestation at 5 mg/kg/day were statistically comparable to vehicle control group.
At 15 mg/kg/day, the mean body weights towards the later part of gestation GD 27 and 29 were statistically significantly lower by -5% when compared to the vehicle control group.

At 30 mg/kg/day, the mean body weights and gains towards the later part of gestation GD 27 and 29 were statistically significantly lower by -6 and -7%, and -282%, respectively when compared to the vehicle control group.
Maternal body weight gain corrected for uterine weight was also statistically significantly lower by -5 and -7% at 15 and 30 mg/kg/day, respectively, compared to the vehicle control group. Corrected body weight gain was statistically significantly lower at 15 and 30 mg/kg/day by -206 and -399%, respectively.

The significant decreases seen in the body weights towards the later part of gestation at 15 and 30 mg/kg/day associated with lower food consumption are considered treatment-related changes.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption during the different days of gestation period at 5 mg/kg/day were statistically comparable to vehicle control group except during GD 9-12 and the reduction was statistically significantly lower by -6%.
Food consumption was significantly lower during GDs 9-12, 12-15, 15-18, 21 24 and 27-29 with a reduction of -5 to -17% at 15 mg/kg/day, and GDs 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 with a reduction of -4 to -61% at 30 mg/kg/day compared to vehicle control group.
The overall food consumption was significantly lower during GDs 6-29 and 0 29 at 15 and 30 mg/kg/day, with a reduction of -9 and -7% and -26 and 22%, respectively compared to the vehicle control group.
The significant decreases seen in the body weights towards the later part of gestation at 15 and 30 mg/kg/day associated with lower food consumption are considered treatment-related changes.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological findings observed at necropsy in the vehicle control and test item treated rabbits including aborted rabbit.
Neuropathological findings:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
There were incidences of abortion of 2, 1, 1, and 3 at 0, 5, 15 and 30 mg/kg/day.
3 females were prematurely sacrificed because of abortion.
Pre- and post-implantation loss:
effects observed, treatment-related
Total litter losses by resorption:
effects observed, treatment-related
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
At 30 mg/kg/day, significant increase in late resorptions (16.65 vs. 3.08% in vehicle control) resulting in higher post-implantation loss (15.59% vs. 6.39% in vehicle control). One rabbit (RBa8646) had complete resorptions (with all late resorptions). These findings were considered treatment-related.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
The maternal parameters comprising mean number of corpora lutea, implantations, early and late resorptions, pre and post-implantation loss, and rabbits with resorptions were statistically comparable between the vehicle control and rabbits treated up to 15 mg/kg/day.
Gross evaluation of the placentas did not reveal any findings in any rabbits at any tested dose levels.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Remarks on result:
other: not determined as only the draft study report was available at submission

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At 15 mg/kg/day, a significant reduction in fetal weight was observed in females (-9%) compared to the respective control value.

At 30 mg/kg, the mean fetal weight of males (-17%), females (-17%) and combined sex (-18%) were significantly lower than the vehicle control group. This is considered secondary to lower maternal body weights and food consumption.
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
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No test-item-related abnormalities were observed during external observation of the fetuses at any of the doses. There was an incidence of a small fetus (anomaly) in one litter (1/141 at 15 mg/kg/day dose group and fore limb moderate flexion at wrist in one fetus (1/124 in one litter). This was considered an incidental finding and was consistent with our historical control.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test-item-related skeletal malformations observed in fetuses of does treated up to 30 mg/kg/day. All skeletal findings were comparable to the concurrent or historical controls.

Following significant skeletal observations were made:

Significantly higher incidences of extra lumbar centra and arch #8 (44.7%, 63/141 fetuses), 13th rib extra bilateral (22.7%, 32/141) and hypoplastic sternum #5 (11.4%, 16/141 fetuses) were found at 15 mg/kg/day. These observations in rabbit fetuses are considered transient and resolve as the organism matures.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no test-item-related visceral malformations observed in fetuses of doe’s treated up to 30 mg/kg/day. Incidences of liver median lobe extra lobation (variation) at 15 mg/kg/day and hypoplastic gallbladder (anomaly) were observed in test-item-treated groups. These findings were in line with the in-house historical control data and considered spontaneous and not related to the test item treatment.
The number of fetal heads subjected to sectioning was 64, 66, 65, and 59 from 0, 5, 15 and 30 mg/kg/day dose groups, respectively. Fetal heads in all dose groups were normal when subjected to Wilson’s razor-blade sectioning
Details on embryotoxic / teratogenic effects:
The litter parameters comprising total number of fetuses, mean litter size, mean fetal weight, and sex ratio were comparable between the vehicle control group and rabbits treated at 5 mg/kg/day.

At 15 mg/kg/day, a significant reduction in fetal weight was observed in females (-9%) compared to the respective control value.

At 30 mg/kg, the mean fetal weight of males (-17%), females (-17%) and combined sex (-18%) were significantly lower than the vehicle control group. This is considered secondary to lower maternal body weights and food consumption.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Remarks on result:
other: not determined as only the draft study report was available at submission

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, oral administration of Sodium mercaptoacetate at 0, 5, 15 and 30 mg/kg/day to pregnant rabbits during GD 6 to 28 resulted in treatment-related hypoactivity at 30 mg/kg/day. The significantly lower maternal body weights towards the later part of gestation at 15 and 30 mg/kg/day were associated with lower food consumption are considered treatment-related non-adverse changes. At 30 mg/kg/day, the percentage of resorptions were higher, which in turn resulted in higher post-implantation loss. The mean fetal weights of males, females, and combined sex were significantly lower, compared to the vehicle control group and considered secondary to lower maternal body weights and food consumption. Gross pathology evaluation of rabbits revealed no abnormality up to the high dose of 30 mg/kg/day. Fetal external, visceral, and skeletal examination revealed no signs of teratogenicity up to 30 mg/kg/day.
Executive summary:

The objective of this study was to evaluate the developmental and maternal toxicity in pregnant female rabbits and developing embryos/fetuses after oral gavage treatment with test item Thiocare®H102 46% in pregnant rabbits from gestation day (GD) 6 to 28. This study provided a rational basis for risk assessment in humans and helped to establish a No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity in rabbits.


 


The dose levels for this study were selected based on the results of the Dose Range Finding study (DRF) study N6282. In the DRF study, dose levels of 0, 25, 50, 75 and 100 mg/kg/day were evaluated in pregnant rabbits (6 rabbits/group) treated with the test item or vehicle from GD 6 to GD 28 at the dose volume of 2 mL/kg body weight. At 25 mg/kg/day, there was reduction in body weight and food consumption. Doses of 50, 75 and 100 mg/kg/day resulted in unscheduled mortalities. Based on the results of the DRF study in pregnant rabbits, considering treatment related deaths at ≥50 mg/kg/day, a high dose of 30 mg/kg/day was selected for the definitive study. A mid dose of 15 mg/kg/day and a low dose of 5 mg/kg/day were selected to provide a graded response to the test item.


 


In the definitive study, 92 mated female rabbits were assigned to four groups. Each group consisted of 23 mated rabbits (G1: vehicle control, G2: low dose, G3: mid dose and G4: high dose). Day 0 of gestation for each individual female rabbit in the study was considered as the day on which mating had occurred. Test item in vehicle (Milli-Q water) was administered at 5, 15 and 30 mg/kg/day at the dose volume of 2 mL/kg body weight for the low, mid, and high dose groups, respectively. The vehicle control group received the vehicle only.


The following parameters and end points were evaluated in this study: Maternal parameters - clinical signs, body weight, body weight gain, food consumption, gross pathology, gravid uterine weight, number of corpora lutea, implantation sites, early and late resorptions, pre and post implantation loss and fetal parameters (total number of live fetuses, fetal weights, sex, and sex ratio). All the fetuses were examined for external malformations. All the live fetuses were examined for visceral and skeletal variations and malformations.


During the conduct of the experiment, homogeneity and active ingredient analyses were carried out from the dose formulation samples collected prior to initiation of treatment and termination of treatment. The results of formulation analyses were within the acceptable limits.


 


Main findings from the study are summarized below:


 


Mortality, clinical signs, and gross necropsy changes:


There were no death in any group.


The treatment resulted in  clinical signs of hypoactivity in 2/23 rabbits and one rabbit showed posture- sitting with head hung down at 30 mg/kg/day. There were incidences of abortions at all the tested doses (two rabbits in vehicle control group, one each in 5 and 15 mg/kg/day and three rabbits at 30 mg/kg/day). There were no gross pathological findings that could be attributed to the test item.


 


Body weight and food consumption:


No effect was observed at 5 mg/kg/day.


The significantly lower maternal body weight and body weight gains observed during later periods of gestation at 15 and 30 mg/kg/day were associated with lower food consumption and are considered treatment-related changes.


 


Maternal developmental parameters:


No significant changes in any maternal developmental toxicity endpoint were observed at 5 and 15 mg/kg/day. At 30 mg/kg/day, the percentage of late resorptions were higher (16.65 vs. 3.08% in vehicle control), which in turn resulted in higher post-implantation loss (15.59% vs. 6.39% in vehicle control). These findings were considered treatment-related.


No gross pathological changes of the placenta were observed in any of the animals.


 


Litter Parameters:


No effect was observed at 5 and 15 mg/kg/day.


At 30 mg/kg/day, the mean fetal weight of males (-17%), females (-17%) and combined sex (-18%) were significantly lower, compared to the vehicle control group and attributed to secondary to lower maternal body weights and food consumption.


At 15 mg/kg/day, a significant reduction in fetal weight was observed in females (-9%) compared to the respective control value.


Fetal examination: The results from external, visceral, and skeletal examinations did not reveal any significant effects that could be attributed to the test item.


 


Conclusion


In conclusion, oral administration of Thiocare®H102 46% at 0, 5, 15 and 30 mg/kg/day to pregnant rabbits during GD 6 to 28 resulted in treatment-related clinical signs of hypoactivity and posture- sitting with head hung down at 30 mg/kg/day. The significantly lower maternal body weights towards the later part of gestation at 15 and 30 mg/kg/day were associated with lower food consumption are considered treatment-related changes.