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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2020-April 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Dioctadecyl disulphide
EC Number:
219-702-5
EC Name:
Dioctadecyl disulphide
Cas Number:
2500-88-1
Molecular formula:
C36H74S2
IUPAC Name:
dioctadecyl disulphide
Constituent 2
Reference substance name:
Di-n-octadecyl disulphide
IUPAC Name:
Di-n-octadecyl disulphide
Constituent 3
Reference substance name:
Hostanox SE 10
IUPAC Name:
Hostanox SE 10

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Age : 10 - 12 weeks at start of treatment
Sex : Females
Body Weight : 211.96 – 319.77 g at start of treatment
Number of Animals : 96 Females
Animals were maintained under the following environmental conditions:
Temperature : 20.1 – 24.4°C
Relative humidity : 49 – 69%
Light/dark cycle (photoperiod) : 12-hour light and 12-hour dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulation analysis was performed on formulation samples prepared on the first day of dose administration. The concentration of Hostanox SE 10 Pills in the dose formulations were in the range between 92.6% - 95.7%, which were within the acceptance criteria (85% - 115%). All the formulations met the acceptance criteria and confirmed that the animals received the intended dose of test item.
Details on mating procedure:
Pregnant females received on a particular day were randomly allocated to G1- G4 groups ensuring minimum differences in mean body weight between the groups. This procedure was continued till each group gets the required number of females(dams). The dams were grouped into four groups (3 test groups, and 1 vehicle control group).
Duration of treatment / exposure:
Dose formulations of vehicle and test item were administered to respective groups by oral route once daily from GD 5 to GD 19 of presumed gestation.
Frequency of treatment:
Once daily
Duration of test:
Gestation day 5 to Gestation day 19
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
High Dose
Dose / conc.:
300 mg/kg bw/day
Remarks:
Mid Dose
Dose / conc.:
100 mg/kg bw/day
Remarks:
Low Dose
No. of animals per sex per dose:
24 presumed pregnant females
Control animals:
yes
yes, concurrent vehicle
Details on study design:
Dose formulations of vehicle and test item were administered to respective groups by oral route once daily from GD 5 to GD 19 of presumed gestation. Dose was administered using a calibrated disposable syringe attached to a 16 and18-gauge stainless steel ball tipped oral gavage needle. Dose volume was maintained at 5 ml/kg b.w. Individual animal dose volumes were calculated based on their most recent body weight. Homogeneity of the dose formulations during sampling and dosing were maintained by constant stirring using a magnetic stirrer.

Examinations

Maternal examinations:
Routine cage-side observations were carried out for all animals at least once a day throughout the study period for general signs of toxicity and twice a day to check morbidity and mortality. Animals that are found dead were weighed and subjected to post-mortem examination.
Individual animal body weights were recorded on gestation days 0, 3, 5, 8, 11, 14, 17 and 20. Adjusted maternal weight was also calculated (i.e., weight after removal of the uterus).
Body weight gain was calculated for gestation days 0 - 3, 3 - 5, 5 - 8, 8 - 11, 11 - 14, 14 - 17 and 17 - 20. Further, the body weight gains for gestation periods (pre-treatment: 0 - 5), (treatment period: 5 - 20) and overall gain during the gestation period (0 - 20) were calculated.
Feed consumption was measured on gestation days 3, 5, 8, 11, 14, 17 and 20.
On gestation day 20, blood (approximately 2 ml) was collected from all surviving dams through retro-orbital plexus in tubes (without anticoagulant) under mild isoflurane anaesthesia. Serum Samples were analysed using an automated analyser, Centaur XP 2273 using Advia Centaur T3 Lite and solid phase reagent, Advia Centaur T4 Lite and solid phase reagent and Advia Centaur TSH kits (competitive immunoassay for T3 and T4, two-site sandwich immunoassay using direct chemiluminometric technology for TSH). The minimum detectable concentrations for T3 was 0.1 ng/mL, T4 was 0.3 µg/dl and TSH was 0.01 µIU/ml.
On gestation Day 20, all the dams were euthanized (Caesarean section) under CO2 asphyxiation and subjected to detailed gross pathological changes in all the visceral organs of the dams. The weight of thyroid along with parathyroid were recorded and histopathology carried out.
Following termination or as soon as possible after death, the uteri were removed and the pregnancy status of the animals ascertained. Uteri that appear non-gravid was examined with Ammonium sulphide staining to confirm the non-pregnant status. The gravid uterus along with cervix was weighed immediately.
The following maternal data was recorded.
• Pregnancy status
• Uterine weight
• No. of corpora lutea
• Total/viable/dead foetuses
• No. of implantation sites
• Total/early/late resorptions
Based on the uterine observations the following parameters were calculated as per formulae mentioned in Appendices 2 & 6.
• Adjusted Maternal Weight
• Relative Uterus Weight
• Pre and Post Implantation Loss
• Percentage live and dead implants
• Percentage live offspring
Ovaries and uterine content:
The uteri were removed and the pregnancy status of the animals ascertained. Uteri that appear non-gravid was examined with Ammonium sulphide staining to confirm the non-pregnant status. The gravid uterus along with cervix was weighed immediately.
The following maternal data was recorded.
• Pregnancy status
• Uterine weight
• No. of corpora lutea
• Total/viable/dead foetuses
• No. of implantation sites
• Total/early/late resorptions
Based on the uterine observations the following parameters were calculated as per formulae mentioned in Appendices 2 & 6.
• Adjusted Maternal Weight
• Relative Uterus Weight
• Pre and Post Implantation Loss
• Percentage live and dead implants
• Percentage live offspring
Blood sampling:
Serum was collected from all surviving animals on gestation day 20. Blood (approximately 2 ml) was collected from all surviving dams through retro-orbital plexus in tubes (without anticoagulant) under mild isoflurane anaesthesia for estimation of TSH, T3 and T4. Collections were carried out within two hours before necropsy and serum was separated by centrifugation at 2500 to 3500 rpm for 5 to 10 minutes at 2 to 8ºC and stored at approximately -70ºC until analysis.
Fetal examinations:
All the foetuses were removed sequentially and were identified by serial numbers as per SOP.
The following litter data was recorded:
• Sex identification
• Body Weight
• Anogenital distance
• Individual foetus and placenta weight (g) (placenta was discorded)
All the foetuses were examined for external malformations.
Approximately half of the live foetuses per litter will be selected. A detailed soft tissue examination will be performed as per in-house SOP, which will include observation of all the organs and structures of the head, neck, thorax and abdomen. Reproductive tract will be examined for signs of altered development. External foetal sex (as determined by gross examination) will be compared with internal (gonadal) sex. In addition, indication of incomplete testicular descent/cryptorchidism will be noted in male foetuses. The heads of foetuses will be decapitated and preserved in Bouin’s fixative for head razor examination.
Foetuses selected for skeletal examination were eviscerated and fixed in alcohol. A detailed examination of the skeleton (bone + cartilage) were performed after staining with alizarin red S and alcian blue. Examination included observation of all the bone structures and cartilage of the head, spine, rib cage, pelvis and limbs.
Statistics:
Statistical comparisons were carried out for all continuous data belonging to the control and treated groups using unpaired Students t test. Statistical significance was evaluated at p ≤ 0.05 and/or p ≤ 0.01. All quantitative data were summarised and expressed as Mean ± SD. Gross pathology data were compiled based on incidences and severity of changes.
Indices:
• Adjusted Maternal Weight
• Relative Uterus Weight
• Pre and Post Implantation Loss
• Percentage live and dead implants
• Percentage live offspring
Historical control data:
Historical Data is provided to allow comparison with concurrent vehicle control.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One dam (Animal No. T106/056) was found dead on GD 16. Gross pathological evaluations revealed presence of test item formulation in the lung. The cause of death was attributed to accidental wrong dosing.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no effects on maternal body weight. All dams belonging to the control and treated groups displayed normal body weight gain throughout the study period. Adjusted maternal weight (after exclusion of uterine weight) of treated dams derived on GD 20 was comparable to control group animals.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significant change was observed in the average daily feed consumption of dams treated at 100, 300 and 1000 mg/kg/day as compared to control group animals.
Endocrine findings:
no effects observed
Description (incidence and severity):
There were no changes in T3, T4 and TSH values attributable to treatment. A statistically significant increase observed in T3 at 1000 mg/kg b.w./day was within normal biological range. Moreover, no correlating histopathological changes in thyroid/parathyroid was observed. The observed change was hence attributed to random biological variation.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The absolute weight of thyroid along with parathyroid of treated animals were comparable to the control animals. Thyroid/parathyroid belonging to all control and treated group animals were found to be normal (within histological limits).
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Statistical significant decrease observed in late resorptions in dams treated at 100 mg/kg and post implantation losses in dams treated at 100 and 300 mg/kg were non dose dependant, within historical range and hence considered as incidental.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
changes in number of pregnant
changes in pregnancy duration
clinical signs
dead fetuses
early or late resorptions
effects on pregnancy duration
endocrine findings
food consumption and compound intake
maternal abnormalities
mortality
necropsy findings
number of abortions
pre and post implantation loss
total litter losses by resorption
other:
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
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
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed
Localisation:
external: cranium
external: ear
external: eye
external: face
external: limb
external: paw
external: tail
external: trunk
external: anogenital distance
external: anus
external: genital tubercle
external: large intestine
external: thorax
external: umbilicus
external: pelvic region
skeletal: skull
skeletal: skull, fontanelles
skeletal: skull sutures
skeletal: clavicle
skeletal: scapule
skeletal: forelimb
skeletal: sternum
skeletal: rib
skeletal: supernumerary rib
skeletal: vertebra
skeletal: pelvic girdle
skeletal: hindlimb
visceral/soft tissue: integumentary
visceral/soft tissue: gastrointestinal tract
visceral/soft tissue: hepatobiliary
visceral/soft tissue: urinary
visceral/soft tissue: cardiovascular
visceral/soft tissue: heamatopoietic
visceral/soft tissue: immune system
visceral/soft tissue: musculoskeletal system
visceral/soft tissue: nervous system
visceral/soft tissue: central nervous system
visceral/soft tissue: peripheral nervous system
visceral/soft tissue: somatic nervous system
visceral/soft tissue: autonomic nervous system
visceral/soft tissue: endocrine system
visceral/soft tissue: respiratory system
visceral/soft tissue: male reproductive system
visceral/soft tissue: female reproductive system
visceral/soft tissue: eye
visceral/soft tissue: ear

Overall developmental toxicity

Key result
Developmental effects observed:
no
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
Pregnant Wistar rats when treated with the test item at 100, 300 and 1000 mg/kg/day did not reveal any maternal and foetal toxicity. No teratogenic effects attributable to treatment were observed. Based on findings, the NOAEL of the test item for maternal and foetal toxicity in pregnant Wistar rats is 1000 mg/kg bw per day under the tested conditions.
Executive summary:

A total of four groups (G1, G3, G3 and G4) of pregnant Wistar rats were used in the study out of which, Group 1 (G1) served as the vehicle control. Group 2 (G2), Group 3 (G3) and Group 4 (G4) were administered the test item at the dose levels of 100, 300 and 1000 mg/kg/day respectively, through oral route, once daily, from gestation days (GD) 5 to 19.

One dam (Animal No. T106/056) was found dead on GD 16. Gross pathological evaluations revealed presence of test item formulation in the lung. The cause of death was attributed to accidental wrong dosing. All other dams survived the entire treatment period and were found to be normal and were free from all visible clinical signs.

There were no effects on maternal body weight and feed consumption. All dams belonging to the control and treated groups displayed normal body weight gain throughout the study period. Adjusted maternal weight (after exclusion of uterine weight) of treated dams derived on GD 20 was comparable to control group animals.

There were no changes in T3, T4 and TSH values attributable to treatment. Gross pathological observations of control and treated dams did not reveal any abnormalities. The absolute weight of thyroid along with parathyroid of treated animals were comparable to the control animals. Thyroid/parathyroid belonging to all control and treated group animals were found to be normal (within histological limits).

No abnormalities were observed in the evaluated maternal parameters viz., mean gravid absolute and relative uterine weights, placental weight, number of corpora lutea, implantations, resorptions and implantation losses in treated (100, 300 and 1000 mg/kg/day) group as compared to the control group.

There were no significant changes in the mean number of male and female foetuses and total number of foetuses in the treated group (100, 300 and 1000 mg/kg/day) as compared to the control group. Foetus weight was comparable between treatment and control groups. No malformations related to treatment were detected during external examination of the pups belonging to the control as well as treated groups.

No abnormalities attributable to treatment were detected in any of the treated and control groups during visceral and head razor examinations.

Foetal skeletal examination did not reveal any treatment related abnormalities in any of the treated or control groups.

Conclusion

Pregnant Wistar rats when treated with the test item at 100, 300 and 1000 mg/kg/day did not reveal any maternal and foetal toxicity. No teratogenic effects attributable to treatment were observed.

Based on findings, the NOAEL of the test item for maternal and foetal toxicity in pregnant Wistar rats is 1000 mg/kg under the tested conditions.