Octadecyl stearate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Reproductive Toxicity:

Based on the data available from different studies, the No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day for reproductive toxicity, when rodents were treated with test material orally by gavage. 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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data of read across substances

Justification for type of information:

Weight of evidence approach based on the data of the read-across chemicals.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

other: As mention below

Principles of method if other than guideline:

WoE report is based on reproductive toxicity studies i.e. WoE-2,WoE-3 and WoE-4.

GLP compliance:

not specified

Limit test:

no

Justification for study design:

No data available

Species:

rat

Strain:

other: 2.Sprague-Dawley 3.Crj: CD(SD) 4.Sprague-Dawley

Details on species / strain selection:

No data available

Sex:

male/female

Details on test animals or test system and environmental conditions:

2.Details on test animal
TEST ANIMALS
- Source: Central Animal Facility (CAF), NIPER, Sector-67, S.A.S. Nagar, 160 062, Punjab, India.

- Age at study initiation: 7 to 8 weeks old

- Weight at study initiation: Male: 205.78-247.70 g, Female : 185.58-235.40 g

- Fasting period before study: No data available
- Housing: Animals were housed in sterilized solid bottom polypropylene cages with stainless steel grill tops with facilities for food, water bottles and bedding of clean paddy husk. The cages were suspended on stainless steel racks. Animals were identified by assigning a unique identification (ID) number written on the tail, also specified on individual cage tag.

- Diet (e.g. ad libitum): Standard laboratory sterile extruded pelleted rodent feed (Batch No. 0001642169) manufactured by Provimi Animal Nutrition India Pvt. Ltd., Bangalore, ad libitum.

- Water (e.g. ad libitum): Potable tap water filtered through Reviva Reverse Osmosis System (water filter cum purifier) of Eureka Forbes in polypropylene bottles with stainless steel sipper tubes. ad libitum.

- Acclimation period: Five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%):30-70 %,
- Air changes (per hr): 25 ± 5 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

3.Details on test animals and env. conditions
TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 8 weeks old
- Weight at study initiation: male 312.1 to 363.7 g, female 205.3 -230.8 g).
- Fasting period before study:
- Housing: individually housed in a wire mesh floor cage (Nihon Cage ), raised, and allowed to take solid feed
(CE - 2, Japan Clea )
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period:7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1 ° C.
- Humidity (%):50 to 65%,
- Air changes (per hr): about 15 times / hour
- Photoperiod (hrs dark / hrs light): a lighting condition set at 12 hours of lighting (7 am to 7 pm)

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

corn oil

Details on exposure:

2.Details on oral exposure
PREPARATION OF DOSING SOLUTIONS: Dose was freshly prepared prior to dosing on each day. Corn oil was used as a vehicle for this study.Test chemical was administered to each rat at the dose levels of 250, 500 and 1000 mg/kg in the dose volume of 5 ml/kg body weight. The chemical was weighed on a weighing balance. Then, it was transferred to calibrated falcon tube. Some quantity of the corn oil was added initially and vortexed. The sufficient quantity of vehicle was added to make up the required volume of dose.


DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available

- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Chemical was insoluble in water, the corn oil was used as vehicle to deliver the desired dose levels as it is also recommended in the toxicological evaluation guidelines.

- Concentration in vehicle: 0, 250, 500 and 1000 mg/kg/day
- Amount of vehicle (if gavage): 5 ml of corn oil
- Lot/batch no. (if required): Batch no. 51
- Purity: No data available

3.Details on exposure
PREPARATION OF DOSING SOLUTIONS:
The test substance was prepared by suspending it in corn oil and used as a test sample
DIET PREPARATION
- Rate of preparation of diet (frequency):No data available
- Mixing appropriate amounts with (Type of food )
- Storage temperature of food: No data available
VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Concentration in vehicle: 0 ,100, 300 and 1,000 mg/kg bw/day
- Amount of vehicle (if gavage): 5ml/kg bw
- Lot/batch no. (if required): (Lot No. V 6 H 2050, Nacalai Tesque ),
- Purity: No data available

4.Details on exposure
PREPARATION OF DOSING SOLUTIONS:
Test material dissolved in corn oil.
DIET PREPARATION
- Rate of preparation of diet (frequency):No data available
- Mixing appropriate amounts with (Type of food )
- Storage temperature of food: No data available
VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Concentration in vehicle: 0 ,250, 500 and 1,000 mg/kg bw/day
- Amount of vehicle (if gavage): No data available

- Lot/batch no. (if required): No data available
- Purity: No data available

Details on mating procedure:

3.- M/F ratio per cage: 1/1
- Length of cohabitation: at the most 14days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy : until proof of pregnancy (formation of vaginal closing or sperm detection in vagina)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

2.The tet chemical (250, 500 and 1000 mg) was weighed on balance and dissolved in 5 ml of corn oil. An aliquot (5µl) was taken from three different layers of the dose preparations and was mixed in toluene to make the final volume 1ml. The concentration of the substence in each layer was calculated using the calibration curve of standard (5 mg / ml toluene) by UV - Visible spectroscopy using Flex Station at 600 nm.

Duration of treatment / exposure:

2.28 days.
Study1;
Male rats :from two weeks prior to mating, during the mating period and, approximately, two weeks post mating (at least 28 days),
female rats :from two weeks prior to mating, during the mating period, gestation period and 3 days after lactation

3.Males: 42 days
Females: from 14 days prior to mating to day 3 of lactation

4.Male rats :from two weeks prior to mating, during the mating period and, approximately, two weeks post mating (at least 28 days),
female rats :from two weeks prior to mating, during the mating period, gestation period and 3 days after lactation

Frequency of treatment:

daily

Details on study schedule:

No data available

Remarks:

2. 0 ,250, 500 and 1,000 mg/kg bw/day

Remarks:

3. 0 ,100, 300 and 1,000 mg/kg bw/day

Remarks:

4. 0 ,250, 500 and 1,000 mg/kg bw/day

No. of animals per sex per dose:

2.Total: 56
0 mg/kg/day: 7 male, 7 female
250 mg/kg/day: 7 male, 7 female
500 mg/kg/day: 7 male, 7 female
1000 mg/kg/day: 7 male, 7 female

3.Total :104
0 (Vehicle): 13 male and 13 female
100 mg/kg/day: 13 male and 13 female
300 mg/kg/day: 13 male and 13 female
1000 mg/kg/day: 13 male and 13 female

4.Total:80
0 mg/kg bw/day:10 male and 10 female
250mg/kg bw/day:10 male and 10 female
500mg/kg bw/day:10 male and 10 female
1000mg/kg bw/day:10 male and 10 female

Control animals:

yes, concurrent vehicle

Details on study design:

No data available

Positive control:

No data available

Parental animals: Observations and examinations:

2.Observations and examinations performed & frequency
CAGE SIDE OBSERVATIONS: Yes
- Time schedule : Twice daily (morning and evening)
- Cage side observations checked in table [No.?] were included. : Viaibility or moribund

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily for behavioral changes or reaction to treatment and detailed clinical signs were recorded weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: On day 1, 8, 15, 22, 28, and day 29 (before schedule sacrifice)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data available
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data available
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data available

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Weekly once during 28 days of treatment.



3.Parental animals observation and examinations
CAGE SIDE OBSERVATIONS: yes

DETAILED CLINICAL OBSERVATIONS:

Time schedule: Daily

BODY WEIGHT: Yes
Time schedule for examinations: For all males, measurements were made on 1, 8, and 15 days of administration 1 (administration start date), 8, 15,
22, 29, 36, 42 days and postmenopausal days and females for all cases,
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes For all males, measurements were made on 1, 8, and 15 days of administration (administration start date), 8, 15,22, 29, 36, 42 days and postmenopausal days and females for all cases,


Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data available
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data: No data available


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

4.Parental animals observation and examinations
CAGE SIDE OBSERVATIONS: yes

DETAILED CLINICAL OBSERVATIONS: Yes

Time schedule:

BODY WEIGHT: Yes
Time schedule for examinations:
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data available

Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data available
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data: No data available


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

OTHER:

Oestrous cyclicity (parental animals):

3.Estrous cycle length and pattern not performed because the test was Conducted by the TG adopted in 1990.

Sperm parameters (parental animals):

3.Sperm examination were not performed because the test was Conducted by the TG adopted in 1990.

Litter observations:

2.Sacrifice and pathology
GROSS PATHOLOGY: Yes
All animals were sacrificed by CO2 asphyxiation. Complete necropsy was carried out on all the animals to score the gross lesions. Tissues were collected from all animals and preserved in 10% formal saline. However, testes, ovaries and uterus were first fixed in Bouin’s fixative for short duration then transferred to 10% formal saline.


HISTOPATHOLOGY: Yes
The required tissues for histology slide preparations were embedded in paraffin wax, five micrometers tissue sections were cut and stained with haematoxylin and eosin and examined for histopathological changes.

Organ examined: Brain, Stomach, Large intestine Small intestine, Liver, Kidneys, Adrenal gland(s), Spleen, Heart, Thymus, Lungs, Testis / Ovary, Uterus, Sciatic nerve Lymph nodes, Peripheral nerve (Sciatic) and Bone marrow were examined.


3.litter size at birth, neonatal death, sex ratio of pups were observed

4.litter size at birth, neonatal death, sex ratio of pups were observed

Postmortem examinations (parental animals):

3.Postmortem examinations (Parent Animal)
SACRIFICE : males were killing under pentobarbital sodium deep anesthesia the next day (day corresponding to 43 days of administration). Females delivered were sacrificed on the fourth day of nursing, females that did not deliver to the 25th day of pregnancy, females who did not copulate were exsanguinated and lethal under pentobarbital sodium anesthesia at the end of the mating period,
GROSS NECROPSY: yes
HISTOPATHOLOGY / ORGAN WEIGHTS: yes
Weights of the heart, liver, kidney, thymus, testis and epididymis were measured for all cases ,the testes and epididymis were fixed and stored in Bouin's solution, and other organs and brain, spleen, adrenal gland and bladder were fixed and fixed in 10% formalin. For these high-dose groups and control groups, paraffin sections were prepared according to a conventional method, and hematoxylineosin staining was performed to perform histopathological examination.

4.Postmortem examinations (Parent Animal)
SACRIFICE :Dams were sacrifice on 3 days after lactation
GROSS NECROPSY: yes
HISTOPATHOLOGY / ORGAN WEIGHTS: yes

Postmortem examinations (offspring):

3.SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

4.SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations
HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Statistics:

2.Statistical analysis was carried out by using Microsoft Excel and IBM SPSS statistics version-20.0. All analyses and comparisons were evaluated at the 5 % level, statistically significant differences (p ≤0.05) indicated by appropriate notation. The focus was to examine the mean differences and their significance between control and low dose group, control and mid dose group and control and high dose group. The statistical decision was taken by preparing the univariant GLM MODEL procedure was used to check the significance between above mentioned groups. For multiple comparisons Turkey’s HSD test was applied.

3.Dunnett’s or Scheffe’s test for continuous data, Chi square test for copulated index and fertility index, and Mann-Whitney U test or Fisher’s test for histopathological examination data.

Reproductive indices:

No data available

Offspring viability indices:

No data available

Clinical signs:

no effects observed

Description (incidence and severity):

2.effects observed, treatment-related
Soft stool, nasal discharge was observed in all male and female treated animals.

When treated with 250 mg/kg, crust around nostrils in male and perineum soiled with fecal matter in female were observed.

In 1000 mg/kg/dose, red crust around nostrils, perineum soiled and soft feces were observed in female rats.

3.no effects observed
In males, no abnormalities in general conditions were observed. In females, there was only one change from day 42 of administration (day of delivery) to the one in 100 mg / kg administration group (administration 42 days: piloerection, blood-like fluid discharge from the vaginal opening; administration 43 to 44 days: coat contamination) But it recovered on day 45 of administration. In other females, no abnormalities in the general condition were observed

4.no effects observed
There were no treatment-related effects on clinical examination was observed

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

2.No mortality was observed in treated animals.

3.no mortality observed
No deaths in male and female was observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

2.When treated with 500 and 1000 mg/kg/day, In male rat, significant increase were observed. In female rats, significant increase in body weight were observed in 1000 mg/kg/day as compare to control.

3.effects observed, non-treatment-related
In male, the body weight gain increased from 8 to 15 days in the 100 mg / kg administration group significantly (p <0.01) compared with the control group, However, in the group administered with 300 mg / kg or more, there was no significant difference between the control group and treated group. In females, there was no significant difference in body weight and body weight gain between control group and treated group before breeding, pregnancy period and nursing period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

2.No change was observed in food consumption of male and female rat of treated groups.

3.no effects observed
There was no significant difference between the control group and treated group for males and female at any time of feeding intake

Food efficiency:

not specified

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

no effects observed

Description (incidence and severity):

2.No change was observed among all treated male rat but in female rat significant decreased was observed on 4th week of 1000 mg/kg/day dose treatment

Ophthalmological findings:

no effects observed

Description (incidence and severity):

2.No abnormalities were observed in all treated dose groups rat.

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

2.In male rats, when treated with 1000 mg/kg/day testosterone, sodium, total proteins and total cholesterol level were significantly increased and SGOT, SGPT were significantly reduced in treated rats.

Creatinine level was significantly reduced in 500 mg/kg/day treated male rats.

Potassium level was significantly increased in 250 mg/kg/day treated male rat.

When treaed with 1000 mg/kg/day, In female rats, significant reduction were observed in sodium, albumin and glucose level and Total Bile Acids, Potassium and SGPT were significant ly increased as compared to control.

When treated with 500 mg/kg/day, Potassium and SGPT significantly increased and glucose, blood urea nitrogen (BUN) and creatinine were significantly decreased in female treated rat.

When treated with 250 mg/kg/day, Potassium, SGPT and estrogen was significantly increased and SGOT were significantly decreased in female treated rat.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

2.Minimal increased were observed in eosinophils of small intestine, collapsed lung, minimal focal fatty change in liver were observed in 1000 mg/kg/day male rats. In female, minimal focal fatty change in liver, minimal reactive spleen and minimal increased in eosinophils of small intestine and mild changes were observed in liver with focal fatty change and reactive spleen was observed in 1000 mg/kg/day dose animals.However, the biological significance of these findings are not related to test chemical.

3.no effects observed
Atrophy of seminiferous tubules was found in 2 animals in the 1000 mg / kg administration group, one on both sides and one on one side, but both were localized and extremely mildly changed. There were no other abnormalities. Sperm granuloma was observed in one of the control group, and there was no abnormality. There were no abnormalities in the ovaries of the unexpanded and infertile cases.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

3.There was no significant difference between the control group and treated group in the mating rate, conception rate, the number of days taken from the start of cohabitation to mating and the number of estrus periods recurred during that period.
An abnormality in labor condition (not treated with placenta) and abnormality in nursing condition (not collecting children, poor protrusion of nipple, poor protrusion of infant, child's fetus) was administered to one of 100 mg / kg administration group, The decrease in body surface temperature) was observed, and all children died on nursing day 2, but no abnormality was observed in other mother animals.
Birth rate was 100% in all administration groups, and no significant difference was observed between gestation period between control group and treated group. No significant difference was observed between the control group and treated group for the number of luteinism, the number of implantation and the implantation rate of pregnant animals.


4.no effects observed
There were no treatment-related changes in mating, fertility and pregnant indices, or in the number of corpora lutea and implantations, pre-implantation loss, post-implantation loss, litter size at birth, neonatal death.

Dose descriptor:

NOAEL

Remarks:

2.

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

water consumption and compound intake

ophthalmological examination

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: No toxic effects on reproductive organ was observed

Dose descriptor:

NOAEL

Remarks:

3.

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

gross pathology

histopathology: non-neoplastic

reproductive performance

Remarks on result:

other: overall no toxic effects on reproductive performance

Dose descriptor:

NOAEL

Remarks:

4.

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

reproductive performance

Remarks on result:

other: overall no effects on reproductive performance

Critical effects observed:

no

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

3.There was no significant difference between the control group and treated group for the number of births, delivery rate, birth rate, fertility rate, and survival rate on 4th day of newborn.

4.no mortality observed
There were no treatment-related changes in litter size at birth, neonatal death, viability ratio at the doses tested

Body weight and weight changes:

no effects observed

Description (incidence and severity):

3.There was no significant difference between the control group and the treated group on body weight at 0 and 4 days of nursing

4.no effects observed
There were no treatment-related changes in body weights of pups on day 0 and day 4 of lactation

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):

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):

3.No abnormalities were found in all pups examined in either the external observation at day 0 or the autopsy performed at day 4 after birth.

4.no effects observed
There were no treatment-related changes in gross findings at the doses tested.

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Dose descriptor:

NOAEL

Remarks:

3.

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:

other: overall no developmental toxicity was observed

Dose descriptor:

NOAEL

Remarks:

4.

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

gross pathology

Remarks on result:

other: No developmental toxic effects observed

Reproductive effects observed:

no

2.

Table: Summary of Body Weights

Treatment group	Time (Days)
	1	8	15	22	28	Terminal day
Males
Control	217.79	247.35	269.99	290.96	304.89	300.87
250 mg/Kg	224.64	254.35	279.94	305.36	318.22	313.79
500 mg/Kg	228.39	256.96	282.73	312.63	327.08	322.00
1000 mg/Kg	233.18*	261.44*	284.33*	312.10*	322.86	319.86
Females
Control	194.50	206.76	217.21	231.38	234.65	231.10
250 mg/Kg	195.28	206.89	215.12	225.92	232.35	229.71
500 mg/Kg	199.97	214.31	224.03	235.82	241.54	238.41
1000 mg/Kg	217.27*	233.49*	241.12*	254.07*	261.27*	257.44*

 * Significant at p < 0.05 in comparison to control group

Table: Summary of Histopathology findings

Gross findings	Dose groups
	Males				Females
	G1	G2	G3	G4	G5	G6	G7	G8
Incidence (No. of Animals with Findings/ No. of Animals Examined)
	1/7	-	-	3/7	2/7	-	-	3/7
Lung
Lung Collapsed	-	-	-	1/7	-	-	-	-
Liver
Focal Fatty change	-	-	-	3/7	1/7	-	-	1/7
Small intestine
Excess Lymphocyetes	1/7	-	-	-	-	-	-	1/7
Spleen
Reactive	-	-	-	-	1/7	-	-	1/7

3.

Table: Body weight of female rats treated orally with test material in the combined repeat dose and reproductive/ developmental toxicity screening test

Dose (mg/kg)	0	100	300	1000
Days of administration (Pre mating period)
1 (init. Wt.)	216.2 ± 6.4 (13)	216.7 ± 7.0 (13)	216.6 ± 6.8 (13)	216.3 ± 6.7 (13)
8	221.4 ± 7.3 (13)	221.9 ± 8.4 (13)	219.5 ± 8.4 (13)	221.4 ± 5.8 (13)
15	233.7 ± 9.6 (13)	232.0 ± 10.6 (13)	234.9 ± 10.9 (13)	231.2 ± 8.8 (13)
Days of pregnancy
0	240.4 ± 8.8 (13)	238.3 ± 13.1 (12)	236.7 ± 9.2 (12)	238.1 ± 10.4 (13)
7	273.8 ± 15.0 (13)	273.5 ± 12.4 (12)	270.9 ± 11.3 (12)	269.3 ± 12.1 (13)
14	312.2 ± 19.7 (13)	310.4 ± 11.4 (12)	309.1 ± 14.5 (12)	301.9 ± 13.4 (13)
20	391.5 ± 24.0 (13)	383.1 ± 17.3 (12)	386.2 ± 18.2 (12)	374.6 ± 16.1 (13)
Days of lactation
0	278.7 ± 16.7 (13)	270.3 ± 21.0 (12)	271.8 ± 20.4 (12)	270.0 ± 24.5 (13)
4	303.2 ± 17.3 (13)	287.3 ± 15.2 (11)	297.0 ± 11.1 (12)	290.7 ± 15.4 (13)

 

Values are expressed as mean ± S. D. In grams

Parenthesis indicates number of animals.

Table: Summary of reproductive performance in parental rats treated orally with test material in the combined repeat dose and reproductive/developmental toxicity screening test

Dose (mg/kg)	0	100	300	1000
No. Of mated pairs	13	13	13	13
No. Of copulated pairs	13	12	13	13
Copulation index^a)	100	92.3	100	100
No. Of pregnant animals	13	12	12	13
Fertility index^b)	100	100	92.3	100
Pairing days until copulation (Mean ± S. D.)	1.5 ± 1.0	2.3 ± 1.2	2.6 ± 3.8	2.5 ± 2.4
Frequency of vaginal estrus (Mean ± S. D.)	1.0 ± 0.0	1.0 ± 0.0	1.1 ± 0.3	1.1 ± 0.3

 

A) Copulation index= (number of copulated pairs/number of mated pairs) X100; %

B) Fertility index= (number of pregnant animals/number of copulated pairs) X100;

 

Table: Summary of development of pups from Dams treated orally with test material in the combined repeat dose and reproductive/developmental toxicity screening test

Dose (mg/kg)	0	100	300	1000
Number of pregnant females	13	12	12	13
Number of pregnant females with pups alive	13	12	12	13
Gestation index^A)	100	100	100	100
Gestation length in days	22.2 ± 0.4 (13)	22.4 ± 0.5 (12)	22.3 ± 0.5 (12)	22.2 ± 0.4 (13)
Number of Corpora lutea	16.6 ± 1.6 (13)	16.7 ± 1.8 (12)	16.8 ± 2.3 (12)	16.2 ± 1.5 (13)
Number of implantation sites	16.1 ± 1.5 (13)	15.8 ± 2.1 (12)	16.0 ± 1.5 (12)	15.2 ± 3.1 (13)
Implantation index^B)	96.9 ± 5.1 (13)	94.8 ± 7.1 (12)	96.1 ± 6.0 (12)	93.1 ± 15.2 (13)
Day 0 of lactation
Number of pups born	15.2 ± 1.6 (13)	14.8 ± 2.2 (12)	15.2 ± 1.5 (12)	14.3 ± 2.7 (13)
Delivery index^C)	94.5 ± 8.0 (13)	93.5 ± 3.9 (12)	94.9 ± 4.3 (12)	94.9 ± 6.2 (13)
Number of pups alive	14.9 ± 1.6 (13)	14.3 ± 2.2 (12)	15.1 ± 1.6 (12)	14.1 ± 2.7 (13)
Birth index^D)	93.1 ± 8.8 (13)	90.7 ± 8.7 (12)	94.3 ± 5.1 (12)	93.5 ± 7.2 (13)
Live Birth index^E)	98.5 ± 2.8 (13)	97.0 ± 8.5 (12)	99.4 ± 2.2	98.5 ± 4.0
Pups in grams
Male	6.1 ± 0.3 (13)	6.3 ± 0.4 (12)	6.2 ± 0.5 (12)	6.2 ± 0.5 (13)
Female	5.9 ± 0.3 (13)	5.9 ± 0.5 (12)	5.9 ± 0.4 (12)	5.9 ± 0.5 (13)
Sex ratio^F)	1.09 ± 0.46 (13)	0.94 ± 0.36 (12)	1.37 ± 0.65 (12)	1.22 ± 1.18 (13)
Day 4 of lactation
Number of pups alive	14.7 ± 1.4 (13)	13.0 ± 4.7 (12)	15.1 ± 1.6 (12)	14.1 ± 2.7 (13)
Viability index^G)	98.6 ± 2.7 (13)	89.4 ± 28.8 (12)	100.0 ± 0.0 (12)	100.0 ± 0.0 (13)
Pup weight in grams
Male	9.6 ± 0.7 (13)	9.6 ± 1.5 (11)	9.9 ± 0.9 (12)	9.7 ± 1.1 (13)
Female	9.4 ± 0.6 (13)	9.1 ± 1.3 (11)	9.5 ± 0.8 (12)	9.4 ± 1.1 (13)

Values expressed as Mean ± S.D.

Parenthesis indicates the number of litters evaluated

a): gestation index= (Number of pregnant females with pups alive/ Number of pregnant females) X100; %

b): Implantation index= (Number of implantation sites / Number of Corpora lutea) x100; %

c): Delivery index= (Number of pups born/ Number of implantation sites) x100; %

d): Birth index= (Number of pups alive on day 0/ Number of implantation sites) X 100; %

e): Live Birth index (Number of pups alive on day 0/ Number of pups born) X 100; %

 f): Sex ratio= (Number of male pups alive on day 0/ Number of female pups alive on day 0)

g): Viability index= (Number of live pups on day 4/ Number of live pups on day 0) X 100; %            

Conclusions:

The No Observed Adverse Effect level (NOAEL) for the test chemical for reproduction toxicity in test animals were considered to be 1000 mg/Kg bw/day.

Executive summary:

Data available for the target chemicals was reviewed to determine the toxic nature of the test chemical. The studies are as mentioned below:

In 28 days repeated dose toxicity study, the effect of test chemical was evaluated in male and female Sprague Dawley rats. The test chemical was administered by oral gavage in the concentration of 0, 250, 500 and 1000 mg/kg/ body weight/day. The results showed no effect on mortality, no changes were in food consumption and ophthalmology. Changes were observed in clinical signs like soft stool, nasal discharge, red crust around nostrils, perineum soiled with fecal matter. Significant decreased were observed in water consumption and locomotor activity of female rat in1000 mg/kg dose group. Body weight was increased significantly in male and female rats. Significant changes were observed in the level of testosterone, sodium, total proteins, total cholesterol, SGOT, SGPT, albumin, Blood urea nitrogen (BUN) and Creatinine when treated with 500 and 1000 mg/kg/day. Significant changes were observed in absolute and relative weight of brain, adrenals, spleen, thymus, epididymides, heart, kidneys, ovaries, uterus and liver in 500 and 1000 mg/kg/day. In addition, minimal to mild gross pathological and histopathological changes were observed in liver, spleen and intestine. However, the biological significance of these findings are not related to test chemical. Therefore, NOAEL was considered to be 1000 mg/kg/day when Sprague-Dawley rat exposed to test chemical orally.

 

In another Combined Repeat Dose and Reproductive / Developmental Toxicity Screening Test of test material were performed on male and female Sprague Dawley rats. The test material was suspended in corn oil in dose concentration0 ,100, 300 and 1,000 mg/kg bw/day and administered via oral gavage route in dose volume 5ml/kg bw. Dose selected on the bases of preliminary test .All the animals were observed for Clinical signs, body weight and food consumption. No deaths in male and female were observed. However, in the group administered with 300 mg / kg or more, there was no significant difference between the control group and treated group. In females, there was no significant difference in body weight and body weight gain between control group and treated group before breeding, pregnancy period and nursing period. There was no significant difference between the control group and treated group for males and female at any time of feeding intake. There was no significant difference between the control group and treated group in the mating rate, conception rate, the number of days taken from the start of cohabitation to mating and the number of estrus periods recurred during that period. An abnormality in labor condition (not treated with placenta) and abnormality in nursing condition (not collecting children, poor protrusion of nipple, poor protrusion of infant, child's fetus) was administered to one of 100 mg / kg administration group,. Atrophy of seminiferous tubules was found in 2 animals in the 1000 mg / kg administration group, one on both sides and one on one side, but both were localized and extremely mildly changed. There were no other abnormalities. Sperm granuloma was observed in one of the control group, and there was no abnormality. There were no abnormalities in the ovaries of the unexpanded and infertile cases. For female In the 100 mg / kg administration group, the actual weight of the kidney decreased significantly (p <0.05) compared to the control group, but not the dose-dependent change. For other organs in male and female, no significant difference was observed between the control group and treated group. Birth rate was 100% in all administration groups, and no significant difference was observed between gestation period between control group and treated group. No significant difference was observed between the control group and treated group for the number of luteinism, the number of implantation and the implantation rate of pregnant animals.There was no significant difference between the control group and treated group for the number of births, delivery rate, birth rate, fertility rate, and survival rate on 4th day of newborn. In addition, no significant difference was observed between the control group and treated group for sex ratio.There was no significant difference between the control group and thetreated grouponbody weight at 0 and 4 days of nursing. Births that showed morphological abnormalities were not observed in the external table observation on thebirthday, necropsy of the dead child, and necropsy on the 4th day of nursing. Hence No Observed Adverse Effect Level (NOAEL) for reproductive and developmental toxicity was considered to be 1000mg/kg bw .When male and femaleSprague Dawley rats were treated with test material orally.

Furthermore,The reproductive and developmental toxicity study of test chemical was performed on male and female Sprague Dawley rats according to OECD TG 421. The test material dissolved in corn oil in dose concentration 0, 250, 500 and 1,000 mg/kg bw/day via oral gavage route. The male rats treated with test material from two weeks prior to mating, during the mating period and, approximately, two weeks post mating (at least 28 days), and to female rats from two weeks prior to mating, during the mating period, gestation period and 3 days after lactation. Ten males and ten females were used in each group. All the animals were observed for clinical signs, mortality and body weight. There were no treatment-related effects on clinical examination, on body weights, necropsy findings or organ weights. There were no treatment-related changes in mating, fertility and pregnant indices, or in the number of corpora lutea and implantations, pre-implantation loss, post-implantation loss, litter size at birth, neonatal death, sex ratio of pups, viability ratio, body weights of pups on day 0 and day 4 of lactation or gross findings at the doses tested. Hence No Observed Adverse Effect Level (NOAEL) for reproductive and developmental toxicity was considered to be 1000mg/kg bw .When male and female Sprague Dawley rats were treated with test chemical orally.

Based on the data available from different studies, NOAEL was considered to be 1000 mg/kg/day for reproductive toxicity, when rodents 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.

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 Klimisch 2 and from prediction report.

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 :

 

Data available for the target chemicals was reviewed to determine the toxic nature of the test chemical. The studies are as mentioned below:

 

In 28 days repeated dose toxicity study, the effect of test chemical was evaluated in male and female Sprague Dawley rats. The test chemical was administered by oral gavage in the concentration of 0, 250, 500 and 1000 mg/kg/ body weight/day. The results showed no effect on mortality, no changes were in food consumption and ophthalmology. Changes were observed in clinical signs like soft stool, nasal discharge, red crust around nostrils, perineum soiled with fecal matter. Significant decreased were observed in water consumption and locomotor activity of female rat in1000 mg/kg dose group. Body weight was increased significantly in male and female rats. Significant changes were observed in the level of testosterone, sodium, total proteins, total cholesterol, SGOT, SGPT, albumin, Blood urea nitrogen (BUN) and Creatinine when treated with 500 and 1000 mg/kg/day. Significant changes were observed in absolute and relative weight of brain, adrenals, spleen, thymus, epididymides, heart, kidneys, ovaries, uterus and liver in 500 and 1000 mg/kg/day. In addition, minimal to mild gross pathological and histopathological changes were observed in liver, spleen and intestine. However, the biological significance of these findings are not related to test chemical. Therefore, NOAEL was considered to be 1000 mg/kg/day when Sprague-Dawley rat exposed to test chemical orally.

 

In another Combined Repeat Dose and Reproductive / Developmental Toxicity Screening Test of test material were performed on male and female Sprague Dawley rats. The test material was suspended in corn oil in dose concentration0 ,100, 300 and 1,000 mg/kg bw/day and administered via oral gavage route in dose volume 5ml/kg bw. Dose selected on the bases of preliminary test .All the animals were observed for Clinical signs, body weight and food consumption. No deaths in male and female were observed. However, in the group administered with 300 mg / kg or more, there was no significant difference between the control group and treated group. In females, there was no significant difference in body weight and body weight gain between control group and treated group before breeding, pregnancy period and nursing period. There was no significant difference between the control group and treated group for males and female at any time of feeding intake. There was no significant difference between the control group and treated group in the mating rate, conception rate, the number of days taken from the start of cohabitation to mating and the number of estrus periods recurred during that period. An abnormality in labor condition (not treated with placenta) and abnormality in nursing condition (not collecting children, poor protrusion of nipple, poor protrusion of infant, child's fetus) was administered to one of 100 mg / kg administration group,. Atrophy of seminiferous tubules was found in 2 animals in the 1000 mg / kg administration group, one on both sides and one on one side, but both were localized and extremely mildly changed. There were no other abnormalities. Sperm granuloma was observed in one of the control group, and there was no abnormality. There were no abnormalities in the ovaries of the unexpanded and infertile cases. For female In the 100 mg / kg administration group, the actual weight of the kidney decreased significantly (p <0.05) compared to the control group, but not the dose-dependent change. For other organs in male and female, no significant difference was observed between the control group and treated group. Birth rate was 100% in all administration groups, and no significant difference was observed between gestation period between control group and treated group. No significant difference was observed between the control group and treated group for the number of luteinism, the number of implantation and the implantation rate of pregnant animals.There was no significant difference between the control group and treated group for the number of births, delivery rate, birth rate, fertility rate, and survival rate on 4th day of newborn. In addition, no significant difference was observed between the control group and treated group for sex ratio.There was no significant difference between the control group and thetreated grouponbody weight at 0 and 4 days of nursing. Births that showed morphological abnormalities were not observed in the external table observation on thebirthday, necropsy of the dead child, and necropsy on the 4th day of nursing. Hence No Observed Adverse Effect Level (NOAEL) for reproductive and developmental toxicity was considered to be 1000mg/kg bw .When male and femaleSprague Dawley rats were treated with test material orally.

 

Furthermore,The reproductive and developmental toxicity study of test chemical was performed on male and female Sprague Dawley rats according to OECD TG 421. The test material dissolved in corn oil in dose concentration 0, 250, 500 and 1,000 mg/kg bw/day via oral gavage route. The male rats treated with test material from two weeks prior to mating, during the mating period and, approximately, two weeks post mating (at least 28 days), and to female rats from two weeks prior to mating, during the mating period, gestation period and 3 days after lactation. Ten males and ten females were used in each group. All the animals were observed for clinical signs, mortality and body weight. There were no treatment-related effects on clinical examination, on body weights, necropsy findings or organ weights. There were no treatment-related changes in mating, fertility and pregnant indices, or in the number of corpora lutea and implantations, pre-implantation loss, post-implantation loss, litter size at birth, neonatal death, sex ratio of pups, viability ratio, body weights of pups on day 0 and day 4 of lactation or gross findings at the doses tested. Hence No Observed Adverse Effect Level (NOAEL) for reproductive and developmental toxicity was considered to be 1000mg/kg bw .When male and female Sprague Dawley rats were treated with test chemical orally.

 

Based on the data available from different studies, NOAEL was considered to be 1000 mg/kg/day for reproductive toxicity, when rodents 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

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Comparing the NOAEL value and effects observed on rodents, with the criteria of CLP regulation the given test chemical is not likely to classify as reproductive toxicant.