Oxacycloheptadec-10-en-2-one

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Reproductive toxicity study:

A reproduction/developmental toxicity screening test was conducted on Sprague-Dawley rats to investigate the possible adverse effect of the test chemical on male and female reproductive performance and early neonatal development after repeated oral dosing. No mortality and clinical sign of toxicity were recorded at any doses during the entire study. There were no changes observed in mean body weight and food consumption at any doses in both sexes during the experimental period.No alteration in absolute and relative weight of testes and epididymites were seen, although the weight of prostate and seminal vesicles with coagulating glands (PSC) were significantly reduced at 500 and 1000 mg/kg. No data on ovary and uterus weight was provided in the study.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, gestation length, live birth index, number of pups, sex ratio and pup survival index at all tested dose groups throughout the lactation period. Pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes were noted in pup weights and ano-genital distances ratio. The retention of nipples in male pups was unaffected as compared to control. No gross external and internal pathological changes were observed in any tested dose groups both in adults and pups.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. There was no treatment related histopathological findings noticed during the microscopic examination of testes, epididymites and ovaries.In conclusion, the repeated administrationof the test chemical exerted no adverse effects on male and female reproductive functions. In males, the test compound significantly reduced the weight of accessory sex glands (PSC) at middle and high doses, which could affect fertility, but the unchanged mating, fertility and gestation indexes showed no functional impairment. Similarly, the test chemicalwas not developmentally toxic or teratogenic as measured endpoints of embryo/fetal viability, growth, or development was unaltered at all doses tested.Hence, the reproductive and developmental NOAEL were considered as 1000 mg/kg body weight/day when male and female SD rats were orally treated during the period of premating, mating, gestation and early lactation.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The data is from the study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

The Reproductive and Developmental Toxicity Screening Test was done to evaluate any toxic effects of the test substance on the ability of reproduction and also on the developmental characteristics of the Sprague Dawley rats.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

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.

IN-LIFE DATES:
From:
To:

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

corn oil

Details on exposure:

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:

Details on mating procedure:

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:

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.

Chromatographic Conditions:
Column: Zorbax Eclipse Plus, C18 250 x 4.6 mm, 5 µm
Flow rate: 1.0 mL/min
Injection volume: 10 µL
Oven temperature: 35ºC
Run time: 10 minutes
Wavelength: 193 nm
Mobile Phase: Acetonitrile
Diluent: Acetonitrile

Duration of treatment / exposure:

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:

The test item or vehicle was administered to animals through oral (gavage) route once daily.

Details on study schedule:

Not Specified

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:

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:

No Specified

Positive control:

No Specified

Parental animals: Observations and examinations:

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). 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 Specified

Litter observations:

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):

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. During necropsy, the males were randomized.

Postmortem examinations (offspring):

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:

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:

Mating Indices, Fertility Indices, Copulatory Indices, Gestation Indices and Implantation Index and Pre and Post implantation Losses .

Offspring viability indices:

Live Birth Indices and Sex Ratio at Birth, Pup Survival Indices and Sex Ratio during lactation and Pre and Postnatal loss.

Clinical signs:

no effects observed

Description (incidence and severity):

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 specified

Mortality:

no mortality observed

Description (incidence):

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):

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 the experimental period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no changes observed in feed consumption at any of the tested dose group animals of either sex during 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:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

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:

not specified

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):

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 (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

reproductive function (oestrous cycle)

reproductive performance

Remarks on result:

other: No effects on reproductive parameters.

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Clinical signs:

no effects observed

Description (incidence and severity):

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

Dermal irritation (if dermal study):

not specified

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 specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

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 (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

gross pathology

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Reproductive effects observed:

not specified

Treatment related:

not specified

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	11		11	11	11
Male Mating Index (%)	91.67		91.67	91.67	91.67
Females showing evidence of copulation (No.)	12		12	12	12
Female Mating Index (%)	100.00		100.00	100.00	100.00
Fertility Indices
Females achieving pregnancy (No.)	12		12	11	11
Female Fertility Index (%)	100.00		100.00	91.67	91.67
Copulatory Indices
Conceiving days 1 to 5 (No.)	6		7	8	8
Conceiving days >6 (No.)	6		5	4	4
Mean Precoital Interval (Days)	5.92		5.42	7.42	8.75
Gestation Indices
Pregnancy≤21 days (No.)	0		0	0	0
Pregnancy = 22 days (No.)	5		9	8	7
Pregnancy≥23 days (No.)	7		3	3	4
Mean Gestation length (Days)	22.58		22.25	22.27	22.36
Gestation Index (%)	100.00		100.00	100.00	100.00
Dams with live young born (No.)	12		12	11	11
Dams with live young at day 4 post-partum (No.)	12		12	11	11
Dams with live young at day 13 post-partum (No.)	12		12	11	11
Implantation Index and Pre and Post implantation Losses
Implants/dam (Mean)	12.58		11.33	11.55	12.18
Corpora luetea/dam (Mean)	13.08		11.83	12.09	12.64
Implantation Index (%)	96.30		95.83	95.34	96.42
Pre-Implantation Loss (%)	3.70		4.17	4.66	3.58
Post-Implantation Loss (%)	0.64		3.31	1.74	1.46
Offspring Viability Indices
Live Birth Indices and Sex Ratio at Birth
Live pups/dam at birth (mean)	12.50		11.00	11.36	12.00
Litter Size (Total No. of pups born/dam) at birth (mean)	12.58		11.33	11.55	12.18
Mean Live Birth Index/dam (%)	99.36		96.69	98.26	98.54
Male Live pups/dam at birth (mean)	6.25		5.33	5.82	5.55
Female Live pups/dam at birth (mean)	6.25		5.67	5.55	6.45
Sex Ratio (male/female)	1.15		1.17	1.10	0.89

Parameters ↓	Group & Dose (mg/kg body weight/day)
	G1 & 0	G2 &250	G3 &500	G4 & 1000
Pup Survival Indices and Sex Ratio during lactation
Mean No. of Pups survived per dam ( LD1 to 4)	12.50	11.00	11.36	12.00
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	1.15	1.17	1.10	0.89
Mean No. of Pups Sacrificed for Blood Collection on LD4	1.42	1.00	1.18	1.55
Mean No. of Pups survived per dam (LD4 to 7)	11.08	10.00	10.18	10.45
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	1.48	1.40	1.47	1.21
Mean No. of Pups survived per dam (LD7 to 13)	11.08	10.00	10.18	10.45
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	1.48	1.40	1.47	1.21
Pre and Postnatal loss
Mean Pre-natal loss (implantations minus live births) (No.)	3.70	4.17	4.66	3.58
Females with 0 (No.)	7	6	6	6
Females with ≥ 1 (No.)	5	6	5	5
Post-natal loss (live births minus alive at post natal day 13)
Females with 0 (No.)	11	10	9	9
Females with ≥ 1 (No.)	1	3	2	2
Litter Observations
Male Pup weight at birth (mean) in gram	5.84	6.07	5.84	6.07
Female Pup weight at birth (mean) in gram	5.54	5.69	5.45	5.74
Male Pup weight on LD4 (mean) in gram	9.54	9.84	9.56	9.74
Female Pup weight on LD4 (mean) in gram	9.10	9.25	9.03	9.61
Male Pup weight on LD7 (mean) in gram	15.10	15.25	15.06	14.97
Female Pup weight on LD7 (mean) in gram	14.83	14.86	14.82	14.91
Male Pup weight on LD13 (mean) in gram	24.85	25.43	25.23	25.07
Female Pup weight on LD13 (mean) in gram	24.80	25.23	25.19	24.84

TABLE 1.        SUMMARYOF CLINICAL SIGNSOF TOXICITY, DETAILED CLINICAL EXAMINATIONAND MORTALITY RECORD

                                                                                                    Refer Appendix 1

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 & F 0	12	N	0/12
G2, M &F250	12	N	0/12
G3, M &F500	12	N	0/12
G4, M &F1000	12	N	0/12

M: Male; N: Normal

 

Conclusions:

The reproductive and developmental No-Observed-Adverse-Effect-Level (NOAEL) for the test item was considered to be 1000 mg/kg/day when administered to male SD rats for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), and to female SD rats for two weeks pre-mating, during mating, gestation and up to lactation day 13, under the experimental conditions described.

Executive summary:

A reproductive and developmental toxicity study of test item was performed on male and female Sprague Dawley rats according to OECDTG 421“Reproduction/Developmental Toxicity Screening Test”.A total of 96 (48 males + 48 females) Sprague Dawley rats were distributed to fourdosegroups, each consisted of 12 males and 12 females.Groups of 12 rats/sex/dose were orally dosed with 0 (vehicle control), 250, 500 and 1000 mg/kg body weight/day. The vehicle and test compound formulations were administered orally by gavage at 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, pregnancyand up to lactation day 13 after which the pups were sacrificed on lactation day 13 and females were sacrificed on lactation day 14 after overnight fasting.All animals were observed for clinical signs, body weight changes and feed consumption. Blood serum was collected from adult males and one pup per litter on lactation day 13 to determine the T4 hormone level. 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 the mating and fertility indexes were calculated for all adult animals. Gross pathology and organ weighing were performed on day 38 for males and on lactation 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. Each litter was examined after delivery (lactation day 1) and the number and sex of pups (litter size), stillbirths and live births were recorded. The pups were observed for clinical signs and external examinations once daily from lactation day 1 to 13.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 alteration in absolute and relative weight of testes and epididymites were seen, although the weight of prostate and seminal vesicles with coagulating glands (PSC) were significantly reduced at 500 and 1000 mg/kg. No data on ovary and uterus weight was provided in the study.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, gestation length, live birth index, number of pups, sex ratio and pup survival index at all tested dose groups throughout the lactation period. Pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes were noted in pup weights and ano-genital distances ratio. The retention of nipples in male pups was unaffected as compared to control. No gross external and internal pathological changes were observed in any tested dose groups both in adults and pups.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. There was no treatment related histopathological findings noticed during the microscopic examination of testes, epididymites and ovaries.In conclusion, the repeated administrationof the test chemical exerted no adverse effects on male and female reproductive functions. In males, the test compound significantly reduced the weight of accessory sex glands (PSC) at middle and high doses, which could affect fertility, but the unchanged mating, fertility and gestation indexes showed no functional impairment. Similarly, the test substancewas not developmentally toxic or teratogenic as measured endpoints of embryo/fetal viability, growth, or development was unaltered at all doses tested.Hence, the reproductive and developmental NOAEL were considered as 1000 mg/kg body weight/day when male and female SD rats were orally treated during the period of pre-mating, mating, gestation and early lactation.

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:

The data is from a Study report and is thus considered as Klimisch 1.

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:

A reproduction/developmental toxicity screening test was conducted on Sprague-Dawley rats to investigate the possible adverse effect of the test chemical on male and female reproductive performance and early neonatal development after repeated oral dosing. No mortality and clinical sign of toxicity were recorded at any doses during the entire study. There were no changes observed in mean body weight and food consumption at any doses in both sexes during the experimental period.No alteration in absolute and relative weight of testes and epididymites were seen, although the weight of prostate and seminal vesicles with coagulating glands (PSC) were significantly reduced at 500 and 1000 mg/kg. No data on ovary and uterus weight was provided in the study.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, gestation length, live birth index, number of pups, sex ratio and pup survival index at all tested dose groups throughout the lactation period. Pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes were noted in pup weights and ano-genital distances ratio. The retention of nipples in male pups was unaffected as compared to control. No gross external and internal pathological changes were observed in any tested dose groups both in adults and pups.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. There was no treatment related histopathological findings noticed during the microscopic examination of testes, epididymites and ovaries.In conclusion, the repeated administrationof the test chemical exerted no adverse effects on male and female reproductive functions. In males, the test compound significantly reduced the weight of accessory sex glands (PSC) at middle and high doses, which could affect fertility, but the unchanged mating, fertility and gestation indexes showed no functional impairment. Similarly, the test chemicalwas not developmentally toxic or teratogenic as measured endpoints of embryo/fetal viability, growth, or development was unaltered at all doses tested.Hence, the reproductive and developmental NOAEL were considered as 1000 mg/kg body weight/day when male and female SD rats were orally treated during the period of premating, mating, gestation and early lactation.

Effects on developmental toxicity

Description of key information

Developmental toxicity study:

A reproduction/developmental toxicity screening test was conducted on Sprague-Dawley rats to investigate the possible adverse effect of the test chemical on male and female reproductive performance and early neonatal development after repeated oral dosing.Groups of 12 rats/sex/dose were orally dosed with 0 (vehicle control), 250, 500 and 1000 mg/kg body weight/day. The vehicle and test compound formulations were administered orally by gavage at the dose volume of 10 ml/kg body weight. 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 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 on lactation 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. Pups did not reveal any clinical signs or external anomalies throughout the lactation period.Dams did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, gestation length, live birth index, number of pups, sex ratio and pup survival index at any dose level throughout the lactation period.No treatment related changes in pup weights and ano-genital distance ratio were noted. The retention of nipples in male pups was unchanged at all doses in comparison to control. There were no gross pathological changes (both external and internal) observed in any tested dose group of adult animals and pups. Hence, the developmental No-Observed-Adverse-Effect-Level (NOAEL) of the test item was considered to be 1000 mg/kg body weight/day 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.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The data is from the study report.

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

The Reproductive and Developmental Toxicity Screening Test was done to evaluate any toxic effects of the test substance on the ability of reproduction and also on the developmental characteristics of the Sprague Dawley rats.

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

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.

IN-LIFE DATES:
From:
To:

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

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:

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.

Chromatographic Conditions:
Column: Zorbax Eclipse Plus, C18 250 x 4.6 mm, 5 µm
Flow rate: 1.0 mL/min
Injection volume: 10 µL
Oven temperature: 35ºC
Run time: 10 minutes
Wavelength: 193 nm
Mobile Phase: Acetonitrile
Diluent: Acetonitrile

Details on mating procedure:

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:

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:

The test item or vehicle was administered to animals through oral (gavage) route once daily.

Duration of test:

approx. 64 days.

Remarks:

0,250,500,1000mg/kg bw/day

No. of animals per sex per dose:

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:

No Specified

Maternal examinations:

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.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: No data
- Skeletal examinations: No data
- Head examinations: No data

Statistics:

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.

Indices:

Mating Indices, Fertility Indices, Copulatory Indices, Gestation Indices and Implantation Index and Pre and Post implantation Losses .Live Birth Indices and Sex Ratio at Birth, Pup Survival Indices and Sex Ratio during lactation and Pre and Postnatal loss.

Historical control data:

No Specified

Clinical signs:

no effects observed

Description (incidence and severity):

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 specified

Mortality:

no mortality observed

Description (incidence):

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):

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 the experimental period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no changes observed in feed consumption at any of the tested dose group animals of either sex during 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:

not specified

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):

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):

There were no gross pathological changes (both external and internal) observed at all the tested dose group adult animals 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):

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:

not specified

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

no changes were noted in pre and post-implantation loss

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

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:

no effects observed

Other effects:

not specified

Dose descriptor:

NOAEL

Remarks:

Maternal developmental

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

mortality

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Remarks on result:

other: No toxic effects were observed

Abnormalities:

not specified

Localisation:

not specified

Fetal body 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.

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

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not specified

External malformations:

no effects observed

Description (incidence and severity):

There were no gross pathological changes ( external ) 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:

no effects observed

Description (incidence and severity):

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

Other effects:

not specified

Dose descriptor:

NOAEL

Remarks:

Developmental

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

external malformations

Remarks on result:

other: No developmental toxic effects were observed

Abnormalities:

not specified

Localisation:

other: not specified

Developmental effects observed:

not specified

Treatment related:

not specified

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	11		11	11	11
Male Mating Index (%)	91.67		91.67	91.67	91.67
Females showing evidence of copulation (No.)	12		12	12	12
Female Mating Index (%)	100.00		100.00	100.00	100.00
Fertility Indices
Females achieving pregnancy (No.)	12		12	11	11
Female Fertility Index (%)	100.00		100.00	91.67	91.67
Copulatory Indices
Conceiving days 1 to 5 (No.)	6		7	8	8
Conceiving days >6 (No.)	6		5	4	4
Mean Precoital Interval (Days)	5.92		5.42	7.42	8.75
Gestation Indices
Pregnancy≤21 days (No.)	0		0	0	0
Pregnancy = 22 days (No.)	5		9	8	7
Pregnancy≥23 days (No.)	7		3	3	4
Mean Gestation length (Days)	22.58		22.25	22.27	22.36
Gestation Index (%)	100.00		100.00	100.00	100.00
Dams with live young born (No.)	12		12	11	11
Dams with live young at day 4 post-partum (No.)	12		12	11	11
Dams with live young at day 13 post-partum (No.)	12		12	11	11
Implantation Index and Pre and Post implantation Losses
Implants/dam (Mean)	12.58		11.33	11.55	12.18
Corpora luetea/dam (Mean)	13.08		11.83	12.09	12.64
Implantation Index (%)	96.30		95.83	95.34	96.42
Pre-Implantation Loss (%)	3.70		4.17	4.66	3.58
Post-Implantation Loss (%)	0.64		3.31	1.74	1.46
Offspring Viability Indices
Live Birth Indices and Sex Ratio at Birth
Live pups/dam at birth (mean)	12.50		11.00	11.36	12.00
Litter Size (Total No. of pups born/dam) at birth (mean)	12.58		11.33	11.55	12.18
Mean Live Birth Index/dam (%)	99.36		96.69	98.26	98.54
Male Live pups/dam at birth (mean)	6.25		5.33	5.82	5.55
Female Live pups/dam at birth (mean)	6.25		5.67	5.55	6.45
Sex Ratio (male/female)	1.15		1.17	1.10	0.89

Parameters ↓	Group & Dose (mg/kg body weight/day)
	G1 & 0	G2 &250	G3 &500	G4 & 1000
Pup Survival Indices and Sex Ratio during lactation
Mean No. of Pups survived per dam ( LD1 to 4)	12.50	11.00	11.36	12.00
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	1.15	1.17	1.10	0.89
Mean No. of Pups Sacrificed for Blood Collection on LD4	1.42	1.00	1.18	1.55
Mean No. of Pups survived per dam (LD4 to 7)	11.08	10.00	10.18	10.45
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	1.48	1.40	1.47	1.21
Mean No. of Pups survived per dam (LD7 to 13)	11.08	10.00	10.18	10.45
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	1.48	1.40	1.47	1.21
Pre and Postnatal loss
Mean Pre-natal loss (implantations minus live births) (No.)	3.70	4.17	4.66	3.58
Females with 0 (No.)	7	6	6	6
Females with ≥ 1 (No.)	5	6	5	5
Post-natal loss (live births minus alive at post natal day 13)
Females with 0 (No.)	11	10	9	9
Females with ≥ 1 (No.)	1	3	2	2
Litter Observations
Male Pup weight at birth (mean) in gram	5.84	6.07	5.84	6.07
Female Pup weight at birth (mean) in gram	5.54	5.69	5.45	5.74
Male Pup weight on LD4 (mean) in gram	9.54	9.84	9.56	9.74
Female Pup weight on LD4 (mean) in gram	9.10	9.25	9.03	9.61
Male Pup weight on LD7 (mean) in gram	15.10	15.25	15.06	14.97
Female Pup weight on LD7 (mean) in gram	14.83	14.86	14.82	14.91
Male Pup weight on LD13 (mean) in gram	24.85	25.43	25.23	25.07
Female Pup weight on LD13 (mean) in gram	24.80	25.23	25.19	24.84

TABLE 1.        SUMMARYOF CLINICAL SIGNSOF TOXICITY, DETAILED CLINICAL EXAMINATIONAND MORTALITY RECORD

                                                                                                    Refer Appendix 1

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 & F 0	12	N	0/12
G2, M &F250	12	N	0/12
G3, M &F500	12	N	0/12
G4, M &F1000	12	N	0/12

M: Male; N: Normal

 

Conclusions:

The reproductive and developmental No-Observed-Adverse-Effect-Level (NOAEL) for the test item was considered to be 1000 mg/kg/day when administered to male SD rats for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), and to female SD rats for two weeks pre-mating, during mating, gestation and up to lactation day 13, under the experimental conditions described.

Executive summary:

A reproductive and developmental toxicity study of test item was performed on male and female Sprague Dawley rats according to OECDTG 421“Reproduction/Developmental Toxicity Screening Test”.A total of 96 (48 males + 48 females) Sprague Dawley rats were distributed to fourdosegroups, each consisted of 12 males and 12 females.Groups of 12 rats/sex/dose were orally dosed with 0 (vehicle control), 250, 500 and 1000 mg/kg body weight/day. The vehicle and test compound formulations were administered orally by gavage at 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, pregnancyand up to lactation day 13 after which the pups were sacrificed on lactation day 13 and females were sacrificed on lactation day 14 after overnight fasting.All animals were observed for clinical signs, body weight changes and feed consumption. Blood serum was collected from adult males and one pup per litter on lactation day 13 to determine the T4 hormone level. 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 the mating and fertility indexes were calculated for all adult animals. Gross pathology and organ weighing were performed on day 38 for males and on lactation 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. Each litter was examined after delivery (lactation day 1) and the number and sex of pups (litter size), stillbirths and live births were recorded. The pups were observed for clinical signs and external examinations once daily from lactation day 1 to 13.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 alteration in absolute and relative weight of testes and epididymites were seen, although the weight of prostate and seminal vesicles with coagulating glands (PSC) were significantly reduced at 500 and 1000 mg/kg. No data on ovary and uterus weight was provided in the study.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, gestation length, live birth index, number of pups, sex ratio and pup survival index at all tested dose groups throughout the lactation period. Pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes were noted in pup weights and ano-genital distances ratio. The retention of nipples in male pups was unaffected as compared to control. No gross external and internal pathological changes were observed in any tested dose groups both in adults and pups.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. There was no treatment related histopathological findings noticed during the microscopic examination of testes, epididymites and ovaries.In conclusion, the repeated administrationof the test chemical exerted no adverse effects on male and female reproductive functions. In males, the test compound significantly reduced the weight of accessory sex glands (PSC) at middle and high doses, which could affect fertility, but the unchanged mating, fertility and gestation indexes showed no functional impairment. Similarly, the test substancewas not developmentally toxic or teratogenic as measured endpoints of embryo/fetal viability, growth, or development was unaltered at all doses tested.Hence, the reproductive and developmental NOAEL were considered as 1000 mg/kg body weight/day when male and female SD rats were orally treated during the period of pre-mating, mating, gestation and early lactation.

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:

The data is from a Study report and is thus considered as Klimisch 1.

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:

A reproduction/developmental toxicity screening test was conducted on Sprague-Dawley rats to investigate the possible adverse effect of the test chemical on male and female reproductive performance and early neonatal development after repeated oral dosing.Groups of 12 rats/sex/dose were orally dosed with 0 (vehicle control), 250, 500 and 1000 mg/kg body weight/day. The vehicle and test compound formulations were administered orally by gavage at the dose volume of 10 ml/kg body weight. 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 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 on lactation 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. Pups did not reveal any clinical signs or external anomalies throughout the lactation period.Dams did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, gestation length, live birth index, number of pups, sex ratio and pup survival index at any dose level throughout the lactation period.No treatment related changes in pup weights and ano-genital distance ratio were noted. The retention of nipples in male pups was unchanged at all doses in comparison to control. There were no gross pathological changes (both external and internal) observed in any tested dose group of adult animals and pups. Hence, the developmental No-Observed-Adverse-Effect-Level (NOAEL) of the test item was considered to be 1000 mg/kg body weight/day 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.

Justification for classification or non-classification

The exposure tothe test chemicalvia oral route did not produce reproductive and developmental toxicity,relevant to humans,among parental animals andtheprogeny and, therefore it isnot classified for reprotoxicityaccording the criteria of the CLP regulations.