Dodecan-5-olide

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Reproductive toxicity study

Based on the data available from different studies, NOAEL for test material was considered to be 1000mg/kg /day for reproductive toxicity, when male and female rats were treated with test material orally. Thus, comparing this value with the criteria of CLP regulation test material is not likely to classify as reproductive toxicant.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Weight of evidence approach based on the available information from various test chemicals.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Principles of method if other than guideline:

WoE report is based on reproductive toxicity studies on rats

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

2.TEST ANIMALS
- Source:In-house bred animals
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) x wks; (F1) x wks: 9 to 11 weeks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g: Males: 240.91 g to 288.02 g
Females: 200.02 g to 232.05 g
- Fasting period before study:
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
i. Pre mating
Two animals of same sex and group per cage were housed.
ii. Mating
During mating, two animals (one male and one female) of same group were housed.
iii. Post mating
After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): Altromin Maintenance diet for rats and mice 1324 manufactured by Altromin Spezialfutter GmbH & Co. KG was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19.1 to 23.6oC
- Humidity (%):40 to 69%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 29 November 2017
To: 09 May 2018

3.TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation (P): 9 to 11 weeks
- Weight at study initiation: (P) Males: 242.03 g to 270.64 g; Females: 211.12 g to 241.96 g;
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
- Diet (e.g. ad libitum): Altromin Maintenance diet for rats and mice 1324 manufactured by Altromin Spezialfutter GmbH & Co. KG was provided ad libitum.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: 19 days (including fourteen days of oestrus cycle evaluation).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 to 23.6 degree celsius.
- Humidity (%): 40 to 69%,
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

nose only

Vehicle:

corn oil

Details on exposure:

2.PREPARATION OF DOSING SOLUTIONS:The test item formulations were freshly prepared before dose administration on each treatment day. The required quantity of test item was weighed into a clean beaker and there by adding little volume of the vehicle into the beaker,mixed well using glass rod and transferred into measuring cylinder. This rinsing procedure was repeated until complete transfer of test item formulation into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get a desired concentration of 25, 50 and 100 mg/mL of test item for low, mid and high dose groups respectively.

DIET PREPARATION
- Rate of preparation of diet (frequency): Not specified
- Mixing appropriate amounts with (Type of food): Not specified
- Storage temperature of food:Not specified

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not miscible with distilled water at a concentration of 100 mg/mL (high dose concentration selected for the study) and forms uniform suspension with corn oil. Hence, corn oil was selected as a vehicle for test item formulation.
- Concentration in vehicle: 0, 250, 500 and 1000 mg/kg bw
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): Batch No: A1708001
- Purity:
3.PREPARATION OF DOSING SOLUTIONS:
Test material dissolved in corn oil
DIET PREPARATION
- Rate of preparation of diet (frequency): Not specified
- Mixing appropriate amounts with (Type of food): Not specified
- Storage temperature of food: Not specified

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test material dissolved in corn oil
- Concentration in vehicle: 0,250,500,1000mg/kg bw
- Amount of vehicle (if gavage): 10mL/kg bw
- Lot/batch no. (if required): A1708001
- Purity: Not specified

Details on mating procedure:

2.- M/F ratio per cage:1:1 ratio
- Length of cohabitation: The female was placed with the same male until pregnancy occurs by evidence of sperm in vaginal smear until two weeks have elapsed.
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: Day ‘0’ pregnancy was confirmed by the presence of sperm in the vaginal smear.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: Not specified
- Further matings after two unsuccessful attempts: [no / yes (explain)]Not specified
- After successful mating each pregnant female was caged (how): females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
- Any other deviations from standard protocol:
3.The males and females were placed in 1:1 ratio. Every morning, the vaginal smear of each female was examined for presence of sperm in the vaginal smear. The female was placed with the same male until pregnancy occurs by evidence of sperm in vaginal smear until two weeks have elapsed. Day ‘0’ pregnancy was confirmed by the presence of sperm in the vaginal smear. In case pairing is unsuccessful, re-mating of females with proven males of the same group was considered for further one week The females confirmed with mating but not littered were sacrificed 25 days after gestation day ‘0’.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and Homogeneity were analysis.
The test item formulations were freshly prepared before dose administration on each treatment day. The required quantity of test item was weighed into a clean beaker and there by adding little volume of the vehicle into the beaker, mixed well using glass rod and transferred into measuring cylinder. This rinsing procedure was repeated until complete transfer of test item formulation into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get a desired concentration of 25, 50 and 100 mg/mL of test item for low, mid and high dose groups respectively.

Duration of treatment / exposure:

2.Male: 37 days
Female: Approx 62 days
3.The male animals were dosed for a total of 37 Days. This duration included two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period.
The female animals were dosed for approx. 64 days. This duration included two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

2.Total: 96
0 mg/kg bw: 12 male, 12 female
250 mg/kg bw: 12 male, 12 female
500 mg/kg bw: 12 male, 12 female
1000 mg/kg bw: 12 male, 12 female
3.Total: 96 animals
0 mg/kg bw: 12 male, 12 female
250 mg/kg bw: 12 male, 12 female
500 mg/kg bw: 12 male, 12 female
1000 mg/kg bw: 12 male, 12 female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses of 0, 250, 500 and 1000 mg/kg body weight for vehicle control, low dose, mid dose and high dose respectively were selected based on the NOAEL of test item Delta Decalactone is >1000 mg/kg body weight in a 28 days repeated dose study (oral) in rats
- Rationale for animal assignment (if not random): The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were distributed to all the groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study did not exceed ± 20% (+2.99% and -11.62% for males and +4.34% and -7.91% for females) of the mean body weight of each sex. The grouping was done one day prior to the initiation of treatment. Body weight of the animals was analyzed statistically for mean body weight to rule out the statistical significant difference between groups within each sex.
- Other:

Parental animals: Observations and examinations:

2.CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked in table [No.?] were included. : mortality and morbidity were examined.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed at receipt, on the first day of dosing, weekly thereafter (varied by -1 day) and at termination. The females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

3.All the animals were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity. All the animals were subjected to detailed clinical examinations on day 1 before treatment and weekly thereafter during treatment. These observations were made outside the home cage and preferably at the same time. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored for two weeks after five days of acclimatization to evaluate its normal oestrus cyclicity (4 to 5 days). Only females with normal oestrus cyclicity were selected for the treatment. Vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa. Oestrus cyclicity was also monitored on the day of sacrifice for females.

Sperm parameters (parental animals):

Not Examined

Litter observations:

2.The number of pups born (dead and live) in a litter, sex, live births and external observations were recorded at birth. Individual body weight of live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 were recorded. The anogenital distance of each pup was measured on postnatal day 4 (lactation day 4) and the ratio of AGD to the cube root of pup body weight was calculated. All survived male pups were examined for appearance of nipples/areolae on postnatal day 13 (lactation day 13). The litter was observed daily in order to note the number of alive, dead and cannibalized pups.
Hormone analysis were also examined.
3.The day of littering was considered as lactation day 1. The number of pups born (dead and live) in a litter, sex, live births and external observations were recorded at birth. Individual body weight of live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 were recorded. The anogenital distance of each pup was measured on postnatal day 4 (lactation day 4) and the ratio of AGD to the cube root of pup body weight was calculated. All survived male pups were examined for appearance of nipples/areolae on postnatal day 13 (lactation day 13). The litter was observed daily in order to note the number of alive, dead and cannibalized pups. All the dead and sacrificed pups were examined and subjected to gross pathological examination. Fertility index for dams, sires and pup live birth index, mean litter size per group, survival index and sex ratio at birth were calculated.

Postmortem examinations (parental animals):

2.Hormone analysis, organ weight and external and internal gross pathological and histopathological examination.
3.The males were sacrificed after completion of 37 days of treatment, females were sacrificed on lactation day 14 and pups were sacrificed on lactation day 13. The animals were fasted overnight, water was provided ad libitum during fasting. The next day, the body weight of all the fasted animals was recorded prior to exsanguination. The vaginal smear of females on the day of necropsy (lactation day 14) was performed and stage of oestrus cycle was recorded. The animals were euthanized using deep CO2 followed by exsanguination and subjected to gross necropsy, external and internal gross pathological examination.

Postmortem examinations (offspring):

2.organ weight and The pups were sacrificed on lactation day 13 and the sacrificed pups and dead pups were examined for gross abnormalities
3.The pups were sacrificed on lactation day 13 and the sacrificed pups and dead pups were examined for gross abnormalities and the findings were recorded. The thyroid along with parathyroid was collected from one male and one female pup per litter on lactation day 13. The thyroid along with parathyroid from adults and pups were preserved in 10% v/v Neutral Buffered Formalin. The thyroid along with parathyroid from adults was weighed post fixation. The histopathological examination of thyroid from pups and adults was not conducted as there were no treatment related effects noted in T4 levels of adult males and lactation day 13 pups.

Statistics:

2.The raw data was subjected to computer statistical processing. The computer printout of the data (in the form of appendix) was verified with the raw data. After verification, the data was subjected to various statistical analyses using SPSS software version 22.
All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05), indicated by the aforementioned tests designated by the superscripts throughout the report as stated below:
* Statistically significant (P<0.05) change than the vehicle control group.
3.The data was subjected to various statistical analyses using SPSS software version 22. Statistical analysis of Body weight, Percent change in body weight, Feed consumption, Copulatory interval, Gestation length, Organ weights, Anogenital distance, Mean pup weight, Live birth Index, Pup survival index was done by One-way ANOVA with Dunnett’s post test. Statistical analysis of Pre/post implantation loss, Pre/post natal loss , No. of resorptions per dam, Corpora lutea per dam, Implantations per dam, No. of live/dead pups/dam, Sex ratio, Litter size was done by Kruskal-Wallis Test. And statistical analysis of Pregnancy rate, No. of litters with/without resorptions, No. of dams with/without live young born, No. of dams with/without dead pups was done by Chi-square test.

Reproductive indices:

Male Mating Index, Male Fertility Index, Female Mating Index, Female Fertility Index, Gestation Index, Live Birth Index (%) per dam and Pup Survival index (%) on lactation day 4 were examined.

Offspring viability indices:

yes, on daye 0 and 13

Clinical signs:

no effects observed

Description (incidence and severity):

2.There were no clinical signs of toxicity observed at any of the tested dose group animals of either sex during the experimental period.
3.There were no clinical signs of toxicity and the detailed clinical examination of animals did not reveal any changes at any of the tested dose group animals of either sex during the experimental period

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

2.no mortality/morbidity observed at any of the tested dose group animals of either sex during the experimental period.
3.There were no mortality/morbidity observed at any of the tested dose group animals of either sex during the experimental period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

2.There were no changes observed in mean body weight and percent change in body weight with respect to day 1 at all the tested group animals of either sex during gestation, lactation and the experimental period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

2.There were no changes observed in feed consumption at any of the tested dose group animals of either sex during during gestation, lactation and the experimental period.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

2.There were no treatment related changes observed in serum T4 levels in adult males at any of the tested dose groups when compared with vehicle control group.
However, statistically significant increase in T4 levels at the high dose group males when compared with vehicle control group males was noted.
This significant variation could be due to two males in 1000 mg/kg bw with a serum T4 level of 142.851 and 140.210 respectively which are significantly more than other animals of same group. Hence, the variations observed in the T4 levels are considered to be incidental.

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

2.No treatment related histopathological findings noticed in the present study.
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen or any cell or stage specificity of testicular findings.
Testes and ovaries did not show any pathological findings/lesions.
3.No treatment related histopathological findings noticed in the present study.
Unilateral, interstitial mononuclear cell (MNC) infiltration in epididymides was observed one male animal from G4 group. This lesion considered as spontaneous, incidental because of lack of consistency, and unilateral in nature.
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen or any cell or stage specificity of testicular findings.
Testes and ovaries did not show any pathological findings/lesions

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Lesions considered spontaneous and incidental were observed in treated and control rats. These lesions consisted of unilateral interstitial mononuclear cell (MNC) infiltration in epididymides.

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no changes observed in the oestrus cyclicity at any of the tested dose group females during pre-mating treatment, mating treatment and on lactation day 14.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

2.There were no changes observed in number of corpora lutea, number of implantations and no changes were noted in pre and post-implantation loss, pre-natal loss, post-natal loss at any of the tested dose group animals when compared with vehicle control group animals. No resorptions were noted at all the dose group animals observed during necropsy.
3.There were no changes observed in the gestation body weight and percent change in gestation body weight during gestation period at any of the tested dose group animals when compared with vehicle control group animals.There were no changes observed in the gestation length, number of pups delivered, sex ratio and live birth index of each litter at any of the tested dose group animals when compared with vehicle control group animals.There were no changes observed in number of corpora lutea, number of implantations and no changes were noted in pre and post-implantation loss, pre-natal loss, post-natal loss at any of the tested dose group animals when compared with vehicle control group animals. No resorptions were noted at all the dose group animals observed during necropsy.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive performance

Remarks on result:

other: No effect observed

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed in any of the pups of tested dose group animals during lactation period.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no treatment related changes observed in the number of pups and pup survival index of each litter at any of the tested dose group animals during lactation period when compared with vehicle control group animals.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes observed in mean pup (male and female) weight on lactation day 1, 4, 7 and 13 at any of the tested dose groups when compared with vehicle control group dams.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no treatment related changes observed in serum T4 levels in lactation day 13 pups at any of the tested dose groups when compared with vehicle control group.

Urinalysis findings:

not specified

Sexual maturation:

no effects observed

Description (incidence and severity):

There were no changes observed in ano-genital distance ratio on lactation day 4 and no occurrences of nipples in male pups of dams at any of the tested dose group litters and vehicle control group litters observed on lactation day 13.

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes (both external and internal) observed at all the tested dose group pups of either sex at all the tested dose groups examined at termination.

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

clinical biochemistry

gross pathology

Remarks on result:

other: No effect observed

Critical effects observed:

no

Reproductive effects observed:

not specified

Treatment related:

not specified

SUMMARY OF THE STUDY

Parameters ↓		Group & Dose (mg/kg body weight/day)
		G1 & 0	G2 & 250	G3 & 500	G4 & 1000
Reproductive Indices
Mating Indices
Pairs started (No.)	12		12	12	12
Males showing evidence of mating (No.) within 14 days*	12		12	10	11
Females showing evidence of copulation (No.)	12		12	12	12
Male Mating Index (%) within 14 days*	100.00		100.00	83.33	91.67
Female Mating Index (%)	100.00		100.00	100.00	100.00
Fertility Indices
Females achieving pregnancy (No.)	12		12	12	12
Male Fertility Index (%)*	100.00		100.00	83.33	91.67
Female Fertility Index (%)	100.00		100.00	100.00	100.00
Copulatory Indices
Conceiving days 1 to 5 (No.)	7		6	6	5
Conceiving days >6 (No.)	5		6	6	7
Mean Precoital Interval (Days)	5.83		6.75	7.83	7.92
Gestation Indices
Pregnancy≤21 days (No.)	1		1	0	0
Pregnancy = 22 days (No.)	4		6	6	4
Pregnancy≥23 days (No.)	7		5	6	8
Mean Gestation length (Days)	22.50		22.33	22.67	22.75
Gestation Index (%)	100.00		100.00	100.00	100.00
Dams with live young born (No.)	12		12	12	12
Dams with live young at day 4 post-partum (No.)	12		12	12	12
Dams with live young at day 13 post-partum (No.)	12		12	12	12
Implantation Index and Pre and Post implantation Losses
Implants/dam (Mean)	10.58		11.75	11.17	11.00
Corpora luetea/dam (Mean)	10.67		11.75	11.25	11.00
Implantation Index (%)	99.17		100.00	99.07	100.00
Pre-Implantation Loss (%)	0.83		0.00	0.93	0.00
Post-Implantation Loss (%)	1.52		0.00	1.39	0.00
Offspring Viability Indices
Live Birth Indices and Sex Ratio at Birth
Live pups/dam at birth (mean)	10.42		11.75	11.00	11.00
Litter Size (Total No. of pups born/dam) at birth (mean)	10.58		11.75	11.00	11.00
Mean Live Birth Index/dam (%)	98.48		100.00	100.00	100.00
Male Live pups/dam at birth (mean)	4.92		5.92	4.83	4.92
Female Live pups/dam at birth (mean)	5.50		5.83	6.17	6.08
Sex Ratio (male/female)	1.23		1.22	0.90	1.05

*: The females were placed with proven male of same test group after 14 days of cohabitation period.

Parameters ↓	Group & Dose (mg/kg body weight/day)
	G1 & 0	G2 & 100	G3 & 300	G4 & 1000
Pup Survival Indices and Sex Ratio during lactation
Mean No. of Pups survived per dam ( LD1 to 4)	10.42	11.75	11.00	11.00
Mean No. of Pups dead per dam ( LD1 to 4)	0.00	0.00	0.00	0.00
Mean Pup Survival Index (%) per dam (LD1 to 4)	100.00	100.00	100.00	100.00
Sex Ratio (male/female) per dam at LD 4	1.23	1.22	0.90	1.05
Mean No. of Pups Sacrificed for Blood Collection on LD4	0.58	1.25	1.08	0.92
Mean No. of Pups survived per dam (LD4 to 7)	9.83	10.50	9.92	10.08
Mean No. of Pups dead per dam (LD4 to 7)	0.00	0.00	0.00	0.00
Mean Pup Survival Index (%) per dam (LD4 to 7)	100.00	100.00	100.00	100.00
Sex Ratio (male/female) per dam at LD 7	1.22	1.55	1.14	1.21
Mean No. of Pups survived per dam (LD7 to 13)	9.83	10.50	9.92	10.08
Mean No. of Pups dead per dam (LD7 to 13)	0.00	0.00	0.00	0.00
Mean Pup Survival Index (%) per dam ( LD7 to 13)	100.00	100.00	100.00	100.00
Sex Ratio (male/female) per dam at LD 13	1.22	1.55	1.14	1.21
Pre and Postnatal loss
Mean Pre-natal (implantations minus live births) (No.)	0.17	0.00	0.17	0.00
Females with 0 (No.)	11	12	11	12
Females with 1 (No.)	1	0	1	0
Post-natal (live births minus alive at post natal day 13)
Females with 0 (No.)	12	12	12	12
Females with ≥ 1 (No.)	0	0	0	0
Litter Observations
Male Pup weight at birth (mean) in gram	7.10	7.00	7.40	7.20
Female Pup weight at birth (mean) in gram	6.68	6.38	6.83	6.39
Male Pup weight on LD4 (mean) in gram	12.07	11.87	11.90	12.28
Female Pup weight on LD4 (mean) in gram	11.20	10.89	10.60	10.61
Male Pup weight on LD7 (mean) in gram	17.10	17.02	17.52	17.43
Female Pup weight on LD7 (mean) in gram	15.88	15.59	15.58	15.22
Male Pup weight on LD13 (mean) in gram	28.06	27.91	28.67	28.17
Female Pup weight on LD13 (mean) in gram	25.98	25.95	25.89	25.60

 SUMMARYOF CLINICAL SIGNSOF TOXICITY, DETAILED CLINICAL EXAMINATIONAND MORTALITY RECORD         

Group, Sex & Dose (mg/kg body weight/day)	No. of Animals	Clinical Signs of Toxicity/ Detailed Clinical Examination	Mortality (No. of Mortality / No. of Animals dosed)
G1, M & 0	12	N	0/12
G2, M & 250	12	N	0/12
G3, M & 500	12	N	0/12
G4, M & 1000	12	N	0/12

M: Male; N: Normal

Group, Sex & Dose (mg/kg body weight/day)	No. of Animals	Clinical Signs of Toxicity/ Detailed Clinical Examination)	Mortality (No. of Mortality / No. of Animals dosed)
G1, F & 0	12	N	0/12
G2, F & 250	12	N	0/12
G3, F & 500	12	N	0/12
G4, F & 1000	12	N	0/12

  SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Epididymes	Testes	Prostate+Seminal vesicles with coagulating glands (PSC)	Thyroid along with parathyroid^#
G1, M & 0	Mean	1.5304	3.4862	3.0974	0.2577
	±SD	0.1461	0.2643	0.3581	0.0253
	n	12	12	12	12
G2, M & 250	Mean	1.5855	3.3497	3.2505	0.2581
	±SD	0.1608	0.2477	0.3081	0.0202
	n	12	12	12	12
G3, M & 500	Mean	1.6304	3.5343	3.1775	0.2575
	±SD	0.1677	0.2791	0.2398	0.0169
	n	12	12	12	12
G4, M & 1000	Mean	1.5877	3.2692	3.1080	0.2476
	±SD	0.1734	0.1988	0.3843	0.0207
	n	12	12	12	12

M: Male;SD: Standard Deviation; n: Number of animals; #: Weighed post fixation

Group, Sex & Dose (mg/kg body weight/day)		Thyroid along with parathyroid^#
G1, F & 0	Mean	0.2341
	±SD	0.0105
	n	11
G2, F & 100	Mean	0.2414
	±SD	0.0113
	n	10
G3, F & 300	Mean	0.2457
	±SD	0.0126
	n	11
G4, F & 1000	Mean	0.2354
	±SD	0.0162
	n	11

SUMMARY OF FASTING BODY WEIGHT (g) AND ORGAN WEIGHT (%) RELATIVE TO FASTING BODY WEIGHT RECORD

Group, Sex & Dose (mg/kg body weight/day)		Fasting Body Weight (g)	Epididymes	Testes	Prostate+Seminal vesicles with coagulating glands (PSC)	Thyroid along with parathyroid
G1, M & 0	Mean	379.21	0.4050	0.9226	0.8197	0.0678
	±SD	24.03	0.0477	0.0933	0.1089	0.0033
	n	12	12	12	12	12
G2, M & 250	Mean	389.42	0.4070	0.8614	0.8382	0.0663
	±SD	23.79	0.0305	0.0584	0.1005	0.0031
	n	12	12	12	12	12
G3, M & 500	Mean	386.93	0.4216	0.9129	0.8228	0.0665
	±SD	16.51	0.0410	0.0506	0.0725	0.0017
	n	12	12	12	12	12
G4, M & 1000	Mean	376.98	0.4211	0.8679	0.8237	0.0656
	±SD	21.53	0.0387	0.0384	0.0824	0.0020
	n	12	12	12	12	12

M: Male;SD: Standard Deviation; n: Number of animals

Group, Sex & Dose (mg/kg body weight/day)		Fasting Body Weight (g)	Thyroid along with parathyroid
G1, F & 0	Mean	264.08	0.0886
	±SD	6.47	0.0024
	n	11	11
G2, F & 250	Mean	272.77	0.0885
	±SD	13.04	0.0020
	n	10	10
G3, F & 500	Mean	274.96	0.0894
	±SD	14.12	0.0014
	n	11	11
G4, F & 1000	Mean	264.70	0.0889
	±SD	16.47	0.0015
	n	11	11

SUMMARY RECORD OF VAGINAL SMEAR EXAMINATION FOR DETERMINATION OF OESTRUS CYCLICITY

Group & Dose (mg/kg body weight/day)	No. of Females	No. of Females with Regular Oestrus Cyclicity during Pre-mating, Mating and on Lactation day 14	No. of Females with Irregular Oestrus Cyclicity during Pre-mating, Mating and on Lactation day 14
G1 & 0	12	12	0
G2 & 250	12	12	0
G3 & 500	12	12	0
G4 & 1000	12	12	0

SUMMARY RECORD OF GESTATION LENGTH (DAYS) AND DELIVERY DATA

Group & Dose (mg/kg body weight/day)		Gestation Length (Days)		Delivery Data
				Litter Size (No.)	Live Pups (No.)				Dead Pups (No.)				Sex Ratio (M/F) at Birth	Live Birth Index (%)
					Total (No.)	Male (No.)	Female (No.)		Total (No.)	Male (No.)	Female (No.)
G1 & 0	Mean	23.00		11.45	11.09	5.09	6.00		0.36	0.36	0.00		1.04	97.35
	±SD	0.89		2.02	1.64	1.70	1.90		0.67	0.67	0.00		0.76	5.05
	n	11		11	11	11	11		11	11	11		11	11
G2 & 250	Mean	23.30		10.70	10.40	4.00	6.40		0.30	0.30	0.00		0.70	96.67
	±SD	0.67		1.64	2.01	1.56	1.78		0.67	0.67	0.00		0.39	8.05
	n	10		10	10	10	10		10	10	10		10	10
G3 & 500	Mean	23.00		12.09	12.00	6.09	5.91		0.09	0.00	0.09		1.23	99.24
	±SD	0.45		1.14	1.18	2.30	1.70		0.30	0.00	0.30		0.87	2.51
	n	11		11	11	11	11		11	11	11		11	11
G4 & 1000	Mean	23.64		11.18	10.82	4.45	6.36		0.36	0.09	0.27		0.76	96.74
	±SD	0.50		2.18	2.27	1.51	1.86		0.67	0.30	0.65		0.34	6.11
	n	11		11	11	11	11		11	11	11		11	11

  M: Male; F: Female;SD: Standard Deviation; n: Number of dams

SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD

Group & Dose (mg/kg body weight/day)		No. of Live Pups At Birth	During LD 1 to 4							Sex Ratio (M/F) at LD 4	No. of Survived Pups during LD 1 to 4	Pup Survival Index (%) LD 1 to 4
			Live Pups (No.)				Dead Pups (No.)
			Total (No.)	Male (No.)	Female (No.)		Total (No.)	Male (No.)	Female (No.)
G1 & 0	Mean	11.09	11.09	5.09	6.00		0.00	0.00	0.00	1.04	11.09	100.00
	±SD	1.64	1.64	1.70	1.90		0.00	0.00	0.00	0.76	1.64	0.00
	n	11	11	11	11		11	11	11	11	11	11
G2 & 250	Mean	10.40	10.40	4.00	6.40		0.00	0.00	0.00	0.70	10.40	100.00
	±SD	2.01	2.01	1.56	1.78		0.00	0.00	0.00	0.39	2.01	0.00
	n	10	10	10	10		10	10	10	10	10	10
G3 & 500	Mean	12.00	12.00	6.09	5.91		0.00	0.00	0.00	1.23	12.00	100.00
	±SD	1.18	1.18	2.30	1.70		0.00	0.00	0.00	0.87	1.18	0.00
	n	11	11	11	11		11	11	11	11	11	11
G4 & 1000	Mean	10.82	10.82	4.45	6.36		0.00	0.00	0.00	0.76	10.82	100.00
	±SD	2.27	2.27	1.51	1.86		0.00	0.00	0.00	0.34	2.27	0.00
	n	11	11	11	11		11	11	11	11	11	11

M: Male: F: Female;SD: Standard Deviation; n: Number of dams; LD: Lactation Day

Group & Dose (mg/kg body weight/day)	Animal No.	Live Pups (No.) on LD 4				Pups Sacrificed for Blood Collection on LD 4 (No.)				Live Pups (No.) on LD 4 after Sacrificed for Blood Collection				During LD 4 to 7								Sex Ratio (M/F) at LD 7	No. of Survived Pups during LD 4 to 7	Pup Survival Index (%) during          LD 4 to 7
														Live Pups (No.)				Dead Pups (No.)
		Male (No.)	Female (No.)	Total (No.)		Male (No.)	Female (No.)	Total (No.)		Male (No.)	Female (No.)	Total (No.)		Male (No.)	Female (No.)	Total (No.)		Male (No.)	Female (No.)	Total (No.)
G1 & 0	Mean	5.09	6.00	11.09		0.00	1.00	1.00		5.09	5.00	10.09		5.09	5.00	10.09		0.00	0.00	0.00		1.30	10.09	100.00
	±SD	1.70	1.90	1.64		0.00	0.89	0.89		1.70	1.67	0.94		1.70	1.67	0.94		0.00	0.00	0.00		1.05	0.94	0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11		11	11	11		11	11	11
G2 & 250	Mean	4.00	6.40	10.40		0.00	0.90	0.90		4.00	5.50	9.50		4.00	5.50	9.50		0.00	0.00	0.00		0.82	9.50	100.00
	±SD	1.56	1.78	2.01		0.00	0.88	0.88		1.56	1.51	1.35		1.56	1.51	1.35		0.00	0.00	0.00		0.46	1.35	0.00
	n	10	10	10		10	10	10		10	10	10		10	10	10		10	10	10		10	10	10
G3 & 500	Mean	6.09	5.91	12.00		0.00	1.64	1.64		6.09	4.27	10.36		6.09	4.27	10.36		0.00	0.00	0.00		2.27	10.36	100.00
	±SD	2.30	1.70	1.18		0.00	0.50	0.50		2.30	1.74	0.92		2.30	1.74	0.92		0.00	0.00	0.00		2.62	0.92	0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11		11	11	11		11	11	11
G4 & 1000	Mean	4.45	6.36	10.82		0.00	0.73	0.73		4.45	5.64	10.09		4.45	5.64	10.09		0.00	0.00	0.00		0.87	10.09	100.00
	±SD	1.51	1.86	2.27		0.00	1.01	1.01		1.51	1.36	1.38		1.51	1.36	1.38		0.00	0.00	0.00		0.44	1.38	0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11		11	11	11		11	11	11

Group & Dose (mg/kg body weight/day)	Animal No.	Live Pups (No.) on LD 7				During LD 7 to 13									Sex Ratio (M/F) at LD 13	No. of Survived Pups during LD 7 to 13	Pup Survival Index (%) during LD 7 to 13
						Live Pups (No.)					Dead Pups (No.)
		Male (No.)	Female (No.)	Total (No.)		Male (No.)	Female (No.)	Total (No.)			Male (No.)	Female (No.)	Total (No.)
G1 & 0	Mean	5.09	5.00	10.09		5.09	5.00	10.09			0.00	0.00	0.00		1.30	10.09	100.00
	±SD	1.70	1.67	0.94		1.70	1.67	0.94			0.00	0.00	0.00		1.05	0.94	0.00
	n	11	11	11		11	11	11			11	11	11		11	11	11
G2 & 250	Mean	4.00	5.50	9.50		4.00	5.50	9.50			0.00	0.00	0.00		0.82	9.50	100.00
	±SD	1.56	1.51	1.35		1.56	1.51	1.35			0.00	0.00	0.00		0.46	1.35	0.00
	n	10	10	10		10	10	10			10	10	10		10	10	10
G3 & 500	Mean	6.09	4.27	10.36		6.09	4.27	10.36			0.00	0.00	0.00		2.27	10.36	100.00
	±SD	2.30	1.74	0.92		2.30	1.74	0.92			0.00	0.00	0.00		2.62	0.92	0.00
	n	11	11	11		11	11	11			11	11	11		11	11	11
G4 & 1000	Mean	4.45	5.64	10.09		4.45	5.64	10.09			0.00	0.00	0.00		0.87	10.09	100.00
	±SD	1.51	1.36	1.38		1.51	1.36	1.38			0.00	0.00	0.00		0.44	1.38	0.00
	n	11	11	11		11	11	11			11	11	11		11	11	11

    SUMMARY OF UTERI OBSERVATIONS RECORD

Group & Dose (mg/kg body weight/day)		No. of Corpora lutea	No. of Implantations	Implantation Index	Pre-Implantation Loss (%)	Post-Implantation Loss (%)	Pre-natal Loss (No.)	Post-natal Loss (%)	Post-natal Loss (No.)	No. of Early Resorptions	No. of Late Resorptions
G1 & 0	Mean	11.73	11.45	98.07	1.93	2.65	0.36	0.00	0.00	0.00	0.00
	±SD	2.33	2.02	3.35	3.35	5.05	0.67	0.00	0.00	0.00	0.00
	n	11	11	11	11	11	11	11	11	11	11
G2 & 250	Mean	10.70	10.70	100.00	0.00	3.33	0.30	0.00	0.00	0.00	0.00
	±SD	1.64	1.64	0.00	0.00	8.05	0.67	0.00	0.00	0.00	0.00
	n	10	10	10	10	10	10	10	10	10	10
G3 & 500	Mean	12.27	12.09	98.59	1.41	0.76	0.09	0.00	0.00	0.00	0.00
	±SD	1.19	1.14	3.14	3.14	2.51	0.30	0.00	0.00	0.00	0.00
	n	11	11	11	11	11	11	11	11	11	11
G4 & 1000	Mean	11.36	11.18	98.78	1.22	3.26	0.36	0.00	0.00	0.00	0.00
	±SD	2.46	2.18	2.72	2.72	6.11	0.67	0.00	0.00	0.00	0.00
	n	11	11	11	11	11	11	11	11	11	11

SD: Standard Deviation; n: Number of animals

 SUMMARY OF SERUM T4 LEVELS (ng/mL) RECORD - MALES

Group, Sex & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1, M & 0	Mean	87.991
	±SD	4.682
	n	12
G2, M & 250	Mean	87.300
	±SD	4.919
	n	12
G3, M & 500	Mean	95.705
	±SD	14.351
	n	12
G4, M & 1000	Mean	104.278*
	±SD	21.130
	n	12

M: Male; SD: Standard Deviation; n: Number of animals

* Statistically significant (P<0.05) change than the vehicle control group.

SUMMARY OF SERUM T4 LEVELS (ng/mL) RECORD - LACTATION DAY 13 PUPS

Group & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1 & 0	Mean	70.752
	±SD	9.415
	n	11
G2 & 250	Mean	62.065
	±SD	8.076
	n	10
G3 & 500	Mean	64.219
	±SD	7.606
	n	11
G4 & 1000	Mean	62.132
	±SD	9.600
	n	11

SD: Standard Deviation; n: Number of Dams (Serum collected from pups per dam pooled for analysis)

Conclusions:

The No Observed Adverse Effect Level (NOAEL) of the test chemical was considered to be 1000 mg/kg body weight when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13, under the experimental conditions

Executive summary:

Data available from different studies were reviewed to determine the reproductive toxicity of testchemical.The studies are as mentioned below:

Study 2

In a Reproduction/Developmental Toxicity Screening Test, Sprague Dawley rats were treated with test chemical at dose levels of 250, 500 and 1000 mg/kg body weight orally by gavage. Males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed). All the tested dose group animals of either sex did not reveal any clinical signs of toxicity and no mortality/morbidity observed. There were no changes observed in mean body weight, percent change in body weight with respect to day 1 and feed consumption at all the tested dose group animals of either sex during the experimental period. No changes were observed in organ weights (both absolute and relative) at all the tested dose group animals. There were no treatment related changes observed in serum T4 levels of adult males and in serum T4 levels of lactation day 13 pups at any of the tested dose groups. Dams did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index at all the tested dose groups throughout the lactation period. Similarly, all pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes in pup weights, ano-genital distance ratio were noted. No occurrences of nipples in male pups at any of the tested dose groups and vehicle control group. In addition, there were no gross pathological changes (both external and internal) observed at all the tested dose group adult animals and pups. There were no treatment related histopathological findings noticed during the microscopic examination. Testes were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all germ layers (cells). In addition this, ovaries did not show any pathological findings/lesions. Therefore, The No Observed Adverse Effect Level (NOAEL) of the test chemical was considered to be 1000 mg/kg body weight when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13, under the experimental conditions.

Study 3

The reproductive and developmental toxicity study of test item was performed on male and female Sprague Dawley rats according to OECD Guideline for Testing of Chemicals, Number 421, “Reproduction/Developmental Toxicity Screening Test”, adopted on 29July 2016. A total of 96 (48 males + 48 females) Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3and G4) consisted of 12 males and 12 females. The animals in G1 group were administered with vehicle[corn oil],animals in G2, G3 and G4groups were administered withtest item at dose levels of250, 500 and 1000 mg/kg body weight for low dose, mid dose and high dose groups respectively.The vehicleand test item formulationswere administered orally by gavageat the dose volume of 10 mL/kg body weight.

Males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed).

All animals were observed for clinical signs once daily, mortality and morbidity twice daily, detailed clinical examination weekly once, body weight and feed consumption weekly once. The serum collected from adult males and lactation day 13 pups representing each per litter was screened for T4 levels.

Females were observed for oestrus cyclicity during pre-mating treatment and mating treatment period and the dams on lactation day 14 prior to sacrifice. The females were observed for copulatory interval and all the adult animals were observed for mating and fertility index. Each litter was examined after delivery (lactation day 1) and the number and sex of pups (litter size), stillbirths (dead pups born on day 1) and live births were recorded. The dams were observed for body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index throughout the lactation period.The pups were observed for clinical signs and external examinations once daily from lactation day 1 to 13. The both male and female pup weights were recorded separately on lactation days 1, 4, 7 and 13. The anogenital distance of each pup was measured on lactation day 4. The male pups were observed for retention of nipples/areolae on lactation day 13. Gross pathology and organ weighing were performed on day 38 for males and onlactation day 14 for dams. Gross pathology was performed on lactation day 4/13 for pups. The number of corpora lutea and implantation sites for dams were recorded during necropsy.

All the tested dose group animals of either sex did not reveal any clinical signs of toxicity and no mortality/morbidity observed. There were no changes observed in mean body weight, percent change in body weight with respect to day 1 and feed consumption at all the tested dosegroup animals of either sex during the experimental period.

No treatment related changes were observed inorgan weights (both absolute and relative) at all the tested dose group animals. There were no treatment related changes observed in serum T4 levels of adult males and in serum T4 levels of lactation day 13 pups at any of the tested dose groups.

Dams did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index at all the tested dose groups throughout the lactation period.

All pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes in pup weights, ano-genital distance ratio were noted. No occurrences of nipples in male pups at any of the tested dose groups and vehicle control group.

There were no gross pathological changes (both external and internal) observed at all the tested dose group adult animals and pups.There were no treatment related histopathological findings noticed during the microscopic examination.Testes were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all germ layers (cells). In addition this, ovaries did not show any pathological findings/lesions.Hence the No Observed Adverse Effect Level (NOAEL) of the test item was consideredto be 1000 mg/kg body weightwhen administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13,under the experimental conditions employed.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Data is Klimicsh 2 and from authoritative database

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive toxicity study

Data available from different studies were reviewed to determine the reproductive toxicity of testchemical.The studies are as mentioned below:

Study 2

In a Reproduction/Developmental Toxicity Screening Test, Sprague Dawley rats were treated with test chemical at dose levels of 250, 500 and 1000 mg/kg body weight orally by gavage. Males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed). All the tested dose group animals of either sex did not reveal any clinical signs of toxicity and no mortality/morbidity observed. There were no changes observed in mean body weight, percent change in body weight with respect to day 1 and feed consumption at all the tested dose group animals of either sex during the experimental period. No changes were observed in organ weights (both absolute and relative) at all the tested dose group animals. There were no treatment related changes observed in serum T4 levels of adult males and in serum T4 levels of lactation day 13 pups at any of the tested dose groups. Dams did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index at all the tested dose groups throughout the lactation period. Similarly, all pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes in pup weights, ano-genital distance ratio were noted. No occurrences of nipples in male pups at any of the tested dose groups and vehicle control group. In addition, there were no gross pathological changes (both external and internal) observed at all the tested dose group adult animals and pups. There were no treatment related histopathological findings noticed during the microscopic examination. Testes were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all germ layers (cells). In addition this, ovaries did not show any pathological findings/lesions. Therefore, The No Observed Adverse Effect Level (NOAEL) of the test chemical was considered to be 1000 mg/kg body weight when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13, under the experimental conditions.

Study 3

The reproductive and developmental toxicity study of test item was performed on male and female Sprague Dawley rats according to OECD Guideline for Testing of Chemicals, Number 421, “Reproduction/Developmental Toxicity Screening Test”, adopted on 29July 2016. A total of 96 (48 males + 48 females) Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3and G4) consisted of 12 males and 12 females. The animals in G1 group were administered with vehicle[corn oil],animals in G2, G3 and G4groups were administered withtest item at dose levels of250, 500 and 1000 mg/kg body weight for low dose, mid dose and high dose groups respectively.The vehicleand test item formulationswere administered orally by gavageat the dose volume of 10 mL/kg body weight.

Males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed).

All animals were observed for clinical signs once daily, mortality and morbidity twice daily, detailed clinical examination weekly once, body weight and feed consumption weekly once. The serum collected from adult males and lactation day 13 pups representing each per litter was screened for T4 levels.

Females were observed for oestrus cyclicity during pre-mating treatment and mating treatment period and the dams on lactation day 14 prior to sacrifice. The females were observed for copulatory interval and all the adult animals were observed for mating and fertility index. Each litter was examined after delivery (lactation day 1) and the number and sex of pups (litter size), stillbirths (dead pups born on day 1) and live births were recorded. The dams were observed for body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index throughout the lactation period.The pups were observed for clinical signs and external examinations once daily from lactation day 1 to 13. The both male and female pup weights were recorded separately on lactation days 1, 4, 7 and 13. The anogenital distance of each pup was measured on lactation day 4. The male pups were observed for retention of nipples/areolae on lactation day 13. Gross pathology and organ weighing were performed on day 38 for males and onlactation day 14 for dams. Gross pathology was performed on lactation day 4/13 for pups. The number of corpora lutea and implantation sites for dams were recorded during necropsy.

All the tested dose group animals of either sex did not reveal any clinical signs of toxicity and no mortality/morbidity observed. There were no changes observed in mean body weight, percent change in body weight with respect to day 1 and feed consumption at all the tested dosegroup animals of either sex during the experimental period.

No treatment related changes were observed inorgan weights (both absolute and relative) at all the tested dose group animals. There were no treatment related changes observed in serum T4 levels of adult males and in serum T4 levels of lactation day 13 pups at any of the tested dose groups.

Dams did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index at all the tested dose groups throughout the lactation period.

All pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes in pup weights, ano-genital distance ratio were noted. No occurrences of nipples in male pups at any of the tested dose groups and vehicle control group.

There were no gross pathological changes (both external and internal) observed at all the tested dose group adult animals and pups.There were no treatment related histopathological findings noticed during the microscopic examination.Testes were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all germ layers (cells). In addition this, ovaries did not show any pathological findings/lesions.Hence the No Observed Adverse Effect Level (NOAEL) of the test item was consideredto be 1000 mg/kg body weightwhen administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13,under the experimental conditions employed.

Based on the data available from different studies, NOAEL for test material was considered to be 1000mg/kg /day for reproductive toxicity, when male and female rats were treated with test material orally. Thus, comparing this value with the criteria of CLP regulation test material is not likely to classify as reproductive toxicant.

Effects on developmental toxicity

Description of key information

Developmental toxicity study

Based on the various studies available for the test chemical were reviewed to determine the developmental toxicity, NOAELfor test chemical was considered to be 1000 mg /kg bw/day .When rats were treated with test chemical orally. Thus, comparing this value with the criteria of CLP regulation test chemical is not likely to classify as reproductive and developmental toxicant.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Weight of evidence approach based on the available information from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Principles of method if other than guideline:

WoE report is based on developmental toxicity studies on rats

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

2.TEST ANIMALS
- Source:In-house bred animals
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) x wks; (F1) x wks: 9 to 11 weeks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g: Males:
240.91 g to 288.02 g
Females: 200.02 g to 232.05 g
- Fasting period before study:
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
i. Pre mating
Two animals of same sex and group per cage were housed.
ii. Mating
During mating, two animals (one male and one female) of same group were housed.
iii. Post mating
After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): Altromin Maintenance diet for rats and mice 1324 manufactured by Altromin Spezi alfutter GmbH & Co. KG was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19.1 to 23.6oC
- Humidity (%):40 to 69%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 29 November 2017
To: 09 May 2018
3.TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation (P): 9 to 11 weeks
- Weight at study initiation: (P) Males: 242.03 g to 270.64 g; Females: 211.12 g to 241.96 g;
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
- Diet (e.g. ad libitum): Altromin Maintenance diet for rats and mice 1324 manufactured by Altromin Spezialfutter GmbH & Co. KG was provided ad libitum.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: 19 days (including fourteen days of oestrus cycle evaluation).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 to 23.6 degree celsius.
- Humidity (%): 40 to 69%,
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

2.PREPARATION OF DOSING SOLUTIONS:The test item formulations were freshly prepared before dose administration on each treatment day. The required quantity of test item was weighed into a clean beaker and there by adding little volume of the vehicle into the beaker, mixed well using glass rod and transferred into measuring cylinder. This rinsing procedure was repeated until complete transfer of test item formulation into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get a desired concentration of 25, 50 and 100 mg/mL of test item for low, mid and high dose groups respectively.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not miscible with distilled
water at a concentration of 100 mg/mL (high dose concentration selected for the study) and forms unif
orm suspension with corn oil. Hence, corn oil was selected as a vehicle for test item formulation.
- Concentration in vehicle: 0, 250, 500 and 1000 mg/kg bw
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): Batch No: A1708001
- Purity:
3.PREPARATION OF DOSING SOLUTIONS:
Test material dissolved in corn oil
DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test material dissolved in corn oil
- Concentration in vehicle: 0,250,500,1000mg/kg bw
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and Homogeneity were analysis.

Details on mating procedure:

2.- M/F ratio per cage:1:1 ratio
- Length of cohabitation: The female was placed with the same male until pregnancy occurs by evidence of sperm in vaginal smear until two weeks have elapsed.
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: Day ‘0’ pregnancy was confirmed by the presence of sperm in the vaginal smear.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: Not specified
- Further matings after two unsuccessful attempts: [no / yes (explain)]Not specified
- After successful mating each pregnant female was caged (how): females were housed individually.Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
- Any other deviations from standard protocol:
3.The males and females were placed in 1:1 ratio. Every morning, the vaginal smear of each female was examined for presence of sperm in the vaginal smear. The female was placed with the same male until pregnancy occurs by evidence of sperm in vaginal smear until two weeks have elapsed. Day ‘0’ pregnancy was confirmed by the presence of sperm in the vaginal smear. In case pairing is unsuccessful, re-mating of females with proven males of the same group was considered for further one week The females confirmed with mating but not littered were sacrificed 25 days after gestation day ‘0’.

Duration of treatment / exposure:

2.Male: 37 days
Female: Approx 62 days
3.The male animals were dosed for a total of 37 Days. This duration included two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period.
The female animals were dosed for approx. 64 days. This duration included two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.

Frequency of treatment:

Daily

Duration of test:

Males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13 and females (dams) were sacrificed on lactation day 14

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

2.Total: 96
0 mg/kg bw: 12 male, 12 female
250 mg/kg bw: 12 male, 12 female
500 mg/kg bw: 12 male, 12 female
1000 mg/kg bw: 12 male, 12 female
3.Total: 96 animals
0 mg/kg bw: 12 male, 12 female
250 mg/kg bw: 12 male, 12 female
500 mg/kg bw: 12 male, 12 female
1000 mg/kg bw: 12 male, 12 female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses of 0, 250, 500 and 1000 mg/kg body weight for vehicle control, low dose, mid dose and high dose respectively were selected based on the NOAEL of test item is >1000 mg/kg body weight in a 28 days repeated dose study (oral) in rats
- Rationale for animal assignment (if not random): The animals were weighed and arranged in ascendin g order of their body weights. These body weight stratified animals were distributed to all the groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study did not exceed ± 20% (+2.99% and -11.62% for males and +4.34% and -7.91% for females) of the mean body weight of each sex. The grouping was done one day prior to the initiation of treatment. Body weight of the animals was analyzed statistically for mean body weight to rule out the statistical significant difference between groups within each sex.
- Other:

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked in table [No.?] were included. : mortality and morbidity were examined
.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily
BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed at receipt, on the first day of dosing, week
ly thereafter (varied by -1 day) and at termination. The females were weighed on gestation days 0, 7, 14
and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/
kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain
data: Yes
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

OTHER:

Ovaries and uterine content:

2& 3The 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:

Fetal examinations:

2&3- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: No data

Statistics:

2&3The raw data was subjected to computer statistical processing. The computer printout of the data (in the form of appendix) was verified with the raw data. After verification, the data was subjected to various statistical analyses using SPSS software version 22.
All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05), indicated by the aforementioned tests designated by the superscripts throughout the report as stated below:
* Statistically significant (P<0.05) change than the vehicle control group.

Indices:

2&3.Male Mating Index, Male Fertility Index, Female Mating Index, Female Fertility Index, Gestation Index, Live Birth Index (%) per dam and Pup Survival index (%) on lactation day 4 were examined.

Clinical signs:

no effects observed

Description (incidence and severity):

2&3.There were no clinical signs of toxicity observed at any of the tested dose group animals of either sex during the experimental period.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

2&3no mortality/morbidity observed at any of the tested dose group animals of either sex during the experimental period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

2&3.There were no changes observed in mean body weight and percent change in body weight with respect to day 1 at all the tested group animals of either sex during gestation, lactation and the experimental period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

2&3.There were no changes observed in feed consumption at any of the tested dose group animals of either sex during during gestation, lactation and the experimental period.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

2.There were no treatment related changes observed in serum T4 levels in adult males at any of the tested dose groups when compared with vehicle control group. However, statistically significant increase in T4 levels at the high dose group males when compared with vehicle control group males was noted.
This significant variation could be due to two males in 1000 mg/kg bw with a serum T4 level of 142.851 and 140.210 respectively which are significantly more than other animals of same group. Hence, the variations observed in the T4 levels are considered to be incidental.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

2.There were no effects observed in absolute and relative organ weights at any of the tested dose group animals of either sex when compared with vehicle control group
3.There were no effects observed in absolute Epididymides, Prostate and Seminal vesicles and coagulating glands, Testes and Thyroid along with parathyroid weights and its relative organ weights with respect to terminal body weights at any of the tested dose group animals of either sex when compared with vehicle control group. However, statistical significant reduction on absolute and relative PSC weight in G3 and G4 group males and reduction in absolute testes weight in G2 group males when compared with control group males. This change is considered as incidental but not treatment related as there were no macroscopic and microscopic findings noted at all these dose groups and the values are within historical range only

Gross pathological findings:

no effects observed

Description (incidence and severity):

2&3.There were no gross pathological changes (both external and internal) observed at all the tested dose group adult animals and pups of either sex at all the tested dose groups examined at termination.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

2.No treatment related histopathological findings noticed in the present study.
Lesions considered spontaneous and incidental were observed in treated and control rats. These lesions consisted of unilateral interstitial mononuclear cell (MNC) infiltration in epididymides.
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen or any cell or stage specificity of testicular findings. Testes and ovaries did not show any pathological findings/lesions.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

2.There were no changes observed in pre and post-implantation loss at any of the tested dose group animals when compared with vehicle control group animals.
3.no changes were noted in pre and post-implantation loss

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no changes observed in loss by resorptions at any of the tested dose group animals when compared with vehicle control group animals.

Early or late resorptions:

no effects observed

Description (incidence and severity):

2.There were no changes observed in early or late resorptions at any of the tested dose group animals when compared with vehicle control group animals.
3.No resorptions were noted at all the dose group animals observed during necropsy

Dead fetuses:

not specified

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

There were no changes observed in the gestation length at any of the tested dose group animals when compared with vehicle control group animals

Changes in number of pregnant:

not specified

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

early or late resorptions

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

maternal abnormalities

mortality

necropsy findings

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Remarks on result:

other: No effect observed

Abnormalities:

not specified

Localisation:

not specified

Description (incidence and severity):

not specified

Fetal body weight changes:

not specified

Description (incidence and severity):

There were no changes observed in mean pup (male and female) weight on lactation day 1, 4, 7 and 13 at any of the tested dose groups when compared with vehicle control group dams.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no treatment related changes observed in the number of pups and pup survival index of each litter at any of the tested dose group animals during lactation period when compared with vehicle control group animals.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No effect on sex ratio of pup were observed as compared to control.

Changes in litter size and weights:

not specified

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

There were no treatment related changes observed in the pup survival index of each litter at any of the tested dose group animals during lactation period of 13 days when compared with vehicle control group animals.

External malformations:

no effects observed

Description (incidence and severity):

There were no gross pathological changes (both external and internal) observed at all the tested dose group pups of either sex at all the tested dose groups examined at termination.

Skeletal malformations:

not specified

Visceral malformations:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Clinical biochemistry findings:
There were no treatment related changes observed in serum T4 levels in lactation day 13 pups at any of the tested dose groups when compared with vehicle control group.

Sexual maturation:
There were no changes observed in ano-genital distance ratio on lactation day 4 and no occurrences of nipples in male pups of dams at any of the tested dose group litters and vehicle control group litters ob served on lactation day 13.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

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 postnatal survival

external malformations

other: ano-genital distance ratio on lactation day 4 and nipples in male pups of dams

Remarks on result:

other: No effect observed

Abnormalities:

not specified

Localisation:

other: not specified

Description (incidence and severity):

not specified

Developmental effects observed:

not specified

Treatment related:

not specified

Conclusions:

The No Observed Adverse Effect Level (NOAEL) of the test chemical was considered to be 1000 mg/kg body weight for P and F1 generation when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13, under the experimental conditions.

Executive summary:

Data available from different studies for test chemicals were reviewed to determine the developmental toxicity of test chemical. The studies are as mentioned below:

Study2

In a experimental study conducted according to OECD 421. Sprague Dawley rats were treated with test chemical at dose levels of 250, 500 and 1000 mg/kg body weight orally by gavage. Males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed). All the tested dose group animals of either sex did not reveal any clinical signs of toxicity and no mortality/morbidity observed. There were no changes observed in mean body weight, percent change in body weight with respect to day 1 and feed consumption at all the tested dose group animals of either sex during the experimental period. No changes were observed in organ weights (both absolute and relative) at all the tested dose group animals. There were no treatment related changes observed in serum T4 levels of adult males and in serum T4 levels of lactation day 13 pups at any of the tested dose groups. Dams did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index at all the tested dose groups throughout the lactation period. Similarly, no developmental effect were observed in all pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes in pup weights, ano-genital distance ratio were noted. No occurrences of nipples in male pups at any of the tested dose groups and vehicle control group. In addition, there were no gross pathological changes (both external and internal) observed at all the tested dose group adult animals and pups. There were no treatment related histopathological findings noticed during the microscopic examination. Testes were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all germ layers (cells). In addition this, ovaries did not show any pathological findings/lesions. Therefore, The No Observed Adverse Effect Level (NOAEL) of the test chemical was considered to be 1000 mg/kg body weight for P and F1 generation when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13, under the experimental conditions.

Study 3

The reproductive and developmental toxicity study of test chemical was performed on male and female Sprague Dawley rats according to OECD Guideline for Testing of Chemicals, Number 421, “Reproduction/Developmental Toxicity Screening Test”, adopted on 29July 2016. A total of 96 (48 males + 48 females) Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3and G4) consisted of 12 males and 12 females. The animals in G1 group were administered with vehicle[corn oil],animals in G2, G3 and G4groups were administered withtest item at dose levels of250, 500 and 1000 mg/kg body weight for low dose, mid dose and high dose groups respectively.The vehicleand test item formulationswere administered orally by gavageat the dose volume of 10 mL/kg body weight.

Males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed).

All animals were observed for clinical signs once daily, mortality and morbidity twice daily, detailed clinical examination weekly once, body weight and feed consumption weekly once. The serum collected from adult males and lactation day 13 pups representing each per litter was screened for T4 levels.

Females were observed for oestrus cyclicity during pre-mating treatment and mating treatment period and the dams on lactation day 14 prior to sacrifice. The females were observed for copulatory interval and all the adult animals were observed for mating and fertility index. Each litter was examined after delivery (lactation day 1) and the number and sex of pups (litter size), stillbirths (dead pups born on day 1) and live births were recorded. The dams were observed for body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index throughout the lactation period.The pups were observed for clinical signs and external examinations once daily from lactation day 1 to 13. The both male and female pup weights were recorded separately on lactation days 1, 4, 7 and 13. The anogenital distance of each pup was measured on lactation day 4. The male pups were observed for retention of nipples/areolae on lactation day 13. Gross pathology and organ weighing were performed on day 38 for males and onlactation day 14 for dams. Gross pathology was performed on lactation day 4/13 for pups. The number of corpora lutea and implantation sites for dams were recorded during necropsy.

All the tested dose group animals of either sex did not reveal any clinical signs of toxicity and no mortality/morbidity observed. There were no changes observed in mean body weight, percent change in body weight with respect to day 1 and feed consumption at all the tested dosegroup animals of either sex during the experimental period.

No treatment related changes were observed inorgan weights (both absolute and relative) at all the tested dose group animals. There were no treatment related changes observed in serum T4 levels of adult males and in serum T4 levels of lactation day 13 pups at any of the tested dose groups.

Dams did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index at all the tested dose groups throughout the lactation period.

All pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes in pup weights, ano-genital distance ratio were noted. No occurrences of nipples in male pups at any of the tested dose groups and vehicle control group.

There were no gross pathological changes (both external and internal) observed at all the tested dose group adult animals and pups.There were no treatment related histopathological findings noticed during the microscopic examination.Testes were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all germ layers (cells). In addition this, ovaries did not show any pathological findings/lesions.Hence the No Observed Adverse Effect Level (NOAEL) of the test chemical was considered to be 1000 mg/kg body weight when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13,under the experimental conditions employed.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Data is Klimicsh 2 and from authoritative database

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental toxicity study

Data available from different studies for test chemicals were reviewed to determine the developmental toxicity of test chemical. The studies are as mentioned below:

Study2

In a experimental study conducted according to OECD 421. Sprague Dawley rats were treated with test chemical at dose levels of 250, 500 and 1000 mg/kg body weight orally by gavage. Males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed). All the tested dose group animals of either sex did not reveal any clinical signs of toxicity and no mortality/morbidity observed. There were no changes observed in mean body weight, percent change in body weight with respect to day 1 and feed consumption at all the tested dose group animals of either sex during the experimental period. No changes were observed in organ weights (both absolute and relative) at all the tested dose group animals. There were no treatment related changes observed in serum T4 levels of adult males and in serum T4 levels of lactation day 13 pups at any of the tested dose groups. Dams did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index at all the tested dose groups throughout the lactation period. Similarly, no developmental effect were observed in all pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes in pup weights, ano-genital distance ratio were noted. No occurrences of nipples in male pups at any of the tested dose groups and vehicle control group. In addition, there were no gross pathological changes (both external and internal) observed at all the tested dose group adult animals and pups. There were no treatment related histopathological findings noticed during the microscopic examination. Testes were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all germ layers (cells). In addition this, ovaries did not show any pathological findings/lesions. Therefore, The No Observed Adverse Effect Level (NOAEL) of the test chemical was considered to be 1000 mg/kg body weight for P and F1 generation when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13, under the experimental conditions.

Study 3

The reproductive and developmental toxicity study of test chemical was performed on male and female Sprague Dawley rats according to OECD Guideline for Testing of Chemicals, Number 421, “Reproduction/Developmental Toxicity Screening Test”, adopted on 29July 2016. A total of 96 (48 males + 48 females) Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3and G4) consisted of 12 males and 12 females. The animals in G1 group were administered with vehicle[corn oil],animals in G2, G3 and G4groups were administered withtest item at dose levels of250, 500 and 1000 mg/kg body weight for low dose, mid dose and high dose groups respectively.The vehicleand test item formulationswere administered orally by gavageat the dose volume of 10 mL/kg body weight.

Males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed).

All animals were observed for clinical signs once daily, mortality and morbidity twice daily, detailed clinical examination weekly once, body weight and feed consumption weekly once. The serum collected from adult males and lactation day 13 pups representing each per litter was screened for T4 levels.

Females were observed for oestrus cyclicity during pre-mating treatment and mating treatment period and the dams on lactation day 14 prior to sacrifice. The females were observed for copulatory interval and all the adult animals were observed for mating and fertility index. Each litter was examined after delivery (lactation day 1) and the number and sex of pups (litter size), stillbirths (dead pups born on day 1) and live births were recorded. The dams were observed for body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index throughout the lactation period.The pups were observed for clinical signs and external examinations once daily from lactation day 1 to 13. The both male and female pup weights were recorded separately on lactation days 1, 4, 7 and 13. The anogenital distance of each pup was measured on lactation day 4. The male pups were observed for retention of nipples/areolae on lactation day 13. Gross pathology and organ weighing were performed on day 38 for males and onlactation day 14 for dams. Gross pathology was performed on lactation day 4/13 for pups. The number of corpora lutea and implantation sites for dams were recorded during necropsy.

All the tested dose group animals of either sex did not reveal any clinical signs of toxicity and no mortality/morbidity observed. There were no changes observed in mean body weight, percent change in body weight with respect to day 1 and feed consumption at all the tested dosegroup animals of either sex during the experimental period.

No treatment related changes were observed inorgan weights (both absolute and relative) at all the tested dose group animals. There were no treatment related changes observed in serum T4 levels of adult males and in serum T4 levels of lactation day 13 pups at any of the tested dose groups.

Dams did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index at all the tested dose groups throughout the lactation period.

All pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes in pup weights, ano-genital distance ratio were noted. No occurrences of nipples in male pups at any of the tested dose groups and vehicle control group.

There were no gross pathological changes (both external and internal) observed at all the tested dose group adult animals and pups.There were no treatment related histopathological findings noticed during the microscopic examination.Testes were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all germ layers (cells). In addition this, ovaries did not show any pathological findings/lesions.Hence the No Observed Adverse Effect Level (NOAEL) of the test chemical was considered to be 1000 mg/kg body weight when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13,under the experimental conditions employed.

Based on the various studies available for the test chemical were reviewed to determine the developmental toxicity, NOAELfor test chemical was considered to be 1000 mg /kg bw/day .When rats were treated with test chemical orally. Thus, comparing this value with the criteria ofCLP regulation testchemical isnot likelyto classify as reproductive and developmental toxicant.

Justification for classification or non-classification

Thus, comparing this value with the criteria of CLP regulation test chemical is not likely to classify as reproductive and developmental toxicant.