Ethylene di(acetate)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 May - 05 Oct 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted 29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Government of India, Department of Science and Technology, New Delhi, India

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethylene di(acetate)

EC Number:

203-881-1

EC Name:

Ethylene di(acetate)

Cas Number:

111-55-7

Molecular formula:

C6H10O4

IUPAC Name:

2-(acetyloxy)ethyl acetate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Expiration date of the batch: 20 Oct 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15 to 25 °C
- Solubility and stability of the test substance in the vehicle were confirmed by analytical methods

Test animals

Species:

rat

Strain:

Wistar

Remarks:

HanTac: WH

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Vivo Bio Tech Ltd., Medak District, Telangana, India
- Age at study initiation: 12 - 13 weeks
- Weight at study initiation: 275.04 - 347.30 g (males) and 173.29 - 209.05 g (females)
- Housing: Pre-mating: two rats of the same sex in sterilised standard polysulfone cages (size 425 x 266 x 185 mm) with a stainless steel top grill having facilities for pelleted food and water supply, steam sterilised clean corn cob as bedding
- Housing: Mating and post-mating: two rats (1 male, 1 female) in sterilised standard polysulfone cages (size 425 x 266 x 185 mm) with a stainless steel top grill having facilities for pelleted food and water supply. After confirming the presence of sperm in the vaginal smear or vaginal plugs, the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. Sterilised nesting material was provided near-term.
- Diet: Teklad Certified (2014C) Global 14% Protein Rodent Maintainance Diet - Pellet, Envigo, Madison, Wisconsin, ad libitum
- Water: Deep bore-well water passed through an activated charcoal filter and exposed to UV rays in 'Aquagard' on-line water filter-cum-purifier (Eureka Forbes Limited, Mumbai, India), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 57 - 67
- Air changes (per hr): 12.3 - 13.6
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 25 Jun 2018 To:13 Aug 2018

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared daily. Required quantities was weighed for each dose level separately into a beaker and a small volume of vehicle (corn oil) was added and stirred using a glass rod till a uniform suspension was obtained. The volume was made up to the mark using the vehicle to yield final concentrations of 20, 60 and 200 mg/mL. The suspensions were mixed well by stirring using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle: Based on a solubility test, the test item forms uniform suspension in corn oil. Hence, corn oil was used as vehicle for dose formulation preparations.
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Until there was evidence of sperm in the vagina smear or a vaginal plug. All females were successfully copulated within five days from the day of cohabitation.
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and test item concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 of the treatment period and during the 2nd month (Day 32) of treatment and analysed. For each set, duplicate samples were drawn from the top, middle and bottom-layers of each preparation and in case of the control duplicate samples from the middle layer were drawn. The test item in the dose formulation was determined using Gas Chromatograph with Flame Ionisation Detection (FID). The test item was used as the analytical standard.
The results were considered acceptable, as the percent agreement of the analysed concentrations were in the range 85% to 115% of the claimed concentrations. Homogeneity of the dose formulation was considered acceptable as the % relative standard deviation from six replicates at each dose level were ≤ 10%.

Duration of treatment / exposure:

Males: 43 days
Females: maximum period of 56 days (14 days pre-mating until lactation day 14 when parental females were sacrificed)

Frequency of treatment:

daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a preliminary range-finding study, in which animals were orally exposed to 50, 200, 500 and 1000 mg/kg bw/day for 14 days. The test item did not cause any toxicological effect on general health at any dose tested in either sex.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to treatment on Day 1 and at weekly intervals thereafter during the treatment period
- In the detailed clinical examination the animals were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements and stereotypies (e.g. excessive grooming, repetitive circling or bizzare behavior like self-mutilation, walking backwards) were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to treatment on Day 1 and at weekly intervals thereafter. Individual body weights of females were recorded in the same manner until cohabitation (till mating confirmation) with males.

FOOD CONSUMPTION: Yes
- Food consumption was calculated by using the food consumed at each interval per cage and dividing by the number of rats per cage and the number if days in the intervening period to determine the food consumption/rat/day. Food consumption was not measured during the cohabitation period. Food consumption of pregnant dams was recorded on GD 7, 14, 20 and on Days 4 and 13 of the lactation period.

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select females with regular 4-5 days cyclicity for the study. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating to determine the Day 0 of pregnancy/treatment-related effects on mating or precoital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal.

Sperm parameters (parental animals):

Tissues collected form all animals in the control and high dose groups were examined microscopically for histopathological changes, including the qualitative assessment of all stages of spermatogenesis and interstitital testicular cell structure in the testis.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality/ cannibalised pups, presence of gross anomalies/malformations, weight gain, physical or behavioral abnormalities, ano-genital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS: Yes, for external deformities; possible cause of death was determined for pups born or found dead.

HORMONE ANALYSIS: Blood samples were collected and serum was separated for the determination of total rodent thyroxin (T4) and rodent thyroid stimulating hormone (TSH) on Day 4 after birth. T4 and TSH were estimated by Enzyme Linked Immuno Sorbent Assay (ELISA).

Postmortem examinations (parental animals):

SACRIFICE: Terminally sacrificed adult animals were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anaesthesia.
- Male animals: All male animals were sacrificed on Day 44.
- Maternal animals: All female were sacrificed on Lactation Day 14. One dam was pre-terminally found dead on gestation Day 19.

GROSS NECROPSY: Yes
Gross necropsy consisted of external and internal examinations. External macroscopical examination paid attention to all organs listed below, as well as the external genitals which were examined for signs of altered development. The following organs were microscopically examined: adrenals, epididymides, ovaries with oviducts, stomach, thymus, thyroid with parathyroids, uterus with cervix, vagina, prostate, seminal vesicles and coagulating glands, testes, levator ani bulbocavernosus muscle complex, cowper’s glands, glans penis.

HISTOPATHOLOGY / ORGAN WEIGHTS: Yes
The following tissues were prepared for microscopic examination and weighed, respectively: adrenals, epididymides, ovaries with oviducts, stomach, thymus, thyroid with parathyroids, uterus with cervix, vagina, prostate, seminal vesicles and coagulating glands, testes, levator ani bulbocavernosus muscle complex, cowper’s glands, glans penis. Samples from all tissues were preserved in 10% neutral buffered formalin, except for the testes.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at day 13 post-partum
- These animals were subjected to post-mortem detailed macroscopic examinations as follows:

GROSS NECROPSY: Yes
Gross necropsy consisted of external and internal examinations. External macroscopical examination paid attention to all organs listed below, as well as the external genitals which were examined for signs of altered development. The following organs were microscopically examined: adrenals, epididymides, ovaries with oviducts, stomach, thymus, thyroid with parathyroids, uterus with cervix, vagina, prostate, seminal vesicles and coagulating glands, testes, levator ani bulbocavernosus muscle complex, cowper’s glands, glans penis.

HISTOPATHOLOGY / ORGAN WEIGHTS: Yes
The following tissues were prepared for microscopic examination and weighed, respectively: adrenals, epididymides, ovaries with oviducts, stomach, thymus, thyroid with parathyroids, uterus with cervix, vagina, prostate, seminal vesicles and coagulating glands, testes, levator ani bulbocavernosus muscle complex, cowper’s glands, glans penis. Samples from all tissues were preserved in 10% neutral buffered formalin, except for the testes.

Statistics:

Built-in statistical tests were used to analyse net body weight gain, food consumption, ano-genital ratio, oestrous cycle length, organ weight ratios, post implantations, pre-coital intervals, mean litte size, sex ratio and gestation length (days), mating, fertility and survival indices. Data was captured using ProvantisTM. Hormonal data was statistically analysed using SYSTAT Statistical package version 12.0. The following tests were applied:
- Levene’s test for homogeneity of variances
- ANOVA using log transformation for one-way analysis of variances (non-optimal/non-normal or heteroschedastic data was transformed prior to ANOVA)
- Dunnett’s t-test for comparison of means between treatment groups and control groups (overall treatment)
- F-test for statistical significance

Reproductive indices:

- Male mating index [%] = Number of males with evidence of mating/Number of males cohabited x 100
- Male fertility index [%] = Number of males siring a litter/impregnated a female/Number of males cohabited x 100
- Female mating index [%] = Number of females mated/Number of females cohabited x 100
- Female fertility index [%] = Number of pregnant females/Number of females used for mating x 100
- Mean number of implantations/group = Total number of implantations/Total number of pregnant animals
- Post implantation loss [%] = (Number of implantations – Number of live pups)/Number of implantations x 100

Offspring viability indices:

- Mean litter size per group = Total number of pups born/Total number of littered animals
- Mean viable litter size = Number of viable pups/Number if females littered
- Live birth index [%] = Number of viable pups born (at first observation)/Total number of pups born (At first observation) x 100
- Day 4 survival index [%] = Number of viable pups on lactation day 4/Number of viable pups born x 100
- Sex ratio [%] = Number of male pups born/Total number of pups born x 100
- Ano-genital Distance Ratio [mm/g1/3] = Ano-genital distance/Cube root of body weight

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

control group: one dead female rat on gestation day 19

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: significantly higher absolute body weight gains in females during days 1-15

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

100 mg/kg bw/day: significantly increased food consumption during days 1-8 in female rats

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

MORTALITY:
One female rat from the control group (Rv117) was found dead on gestation day 19.

BODY WEIGHT AND WEIGHT CHANGES:
The mean body weights and body weight gains were unaffected throughout the treatment period in males and two weeks pre-mating period in females at all the tested doses when compared to vehicle control group. The absolute body weight gains (Day 1-15) were significantly higher in females at 1000 mg/kg bw/day. This was considered toxicologically not significant, as there were no significant differences observed in weekly mean body weights and food consumption.

FOOD CONSUMPTIONS:
The food consumption was not altered throughout the treatment period when compared to the vehicle control. An incidence of statistically significant increase in the food consumption was observed during days 1-8 in the 100 mg/kg bw/day dose group females and it was not related to treatment.

ESTROUS CYCLE:
The calculated mean oestrous cycle length in treated groups was not significantly different from those of the vehicle control group.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1: Summary of detailed clinical examination, clinical signs and mortality

Observation Type: All Types From Day 1 (Start) to 44 (End)		Male				Female
	0	100	300	1000	0	100	300	1000
	mg/kg bwt/day				mg/kg bwt/day
Normal	10	10	10	10	10	10	10	10
Dead					1	0	0	0
Total litter loss					0	0	1	1
Not littered					0	1	0	0

Table 2: Summary of body weight and body weight gains

Day(s) Relative to Start Date			Males				Females
			0	100	300	1000	0	100	300	1000
			mg/kg bw/day				mg/kg bw/day
Body Weight (g)	1 [a]	Mean	308.72	306.35	305.3	306.54	194.16	193.28	193.83	194.39
		SD	22.61	19.07	18.62	18.75	9.09	10.6	10.9	9.99
		N	10	10	10	10	10	10	10	10
	8 [a]	Mean	320.57	319.68	316.24	318.3	194.76	198.37	199.22	200.97
		SD	23.76	21.81	19.94	21.85	9.95	9.77	13.55	11.99
		N	10	10	10	10	10	10	10	10
	15 [a]	Mean	332.21	331.14	323.48	330.4	200.12	203.82	203.3	206.06
		SD	25.87	22.91	25.32	24.6	11.21	9.43	14.96	13.48
		N	10	10	10	10	10	10	10	10
	22 [a]	Mean	338.67	339	331.85	336.39
		SD	26.26	24.07	26.8	27.52
		N	10	10	10	10
	29 [a]	Mean	347.19	347.84	339.79	344.54
		SD	29.72	24.62	29.13	29.91
		N	10	10	10	10
	36 [a]	Mean	356.25	356.68	351.77	350.89
		SD	31.5	26.76	30.05	28.94
		N	10	10	10	10
	43 [a]	Mean	362.23	366.9	364.88	360.56
		SD	29.12	28.81	30.62	29.93
		N	10	10	10	10
Absolute Weight Gain (g)	1 → 43 [a]	Mean	53.51	60.55	59.58	54.02	5.97	10.54	9.47	11.67*
		SD	9.51	12.84	21.93	16.9	4.89	3.73	5.32	5.64
		N	10	10	10	10	10	10	10	10

[a] ANOVA and Dunnett test (Log)

* p < 0.05

Table 3: Summary of food consumption (g/rat/day)

Day(s) Relative to Start Date		Males				Females
		0	100	300	1000	0	100	300	1000
		mg/kg bw/day				mg/kg bw/day
1 → 8	Mean	16.8	16.92	16.13	17.17	12.3	13.82*	12.63	13.42
	SD	0.57	1.58	0.83	1.24	0.84	1.48	1.17	0.9
	N	10	10	10	10	10	10	10	10
8 → 15	Mean	15.67	16.07	14.82	16.43	11.94	12.7	12.52	12.68
	SD	0.56	1.72	1.27	1.25	0.74	1.02	1.24	0.95
	N	10	10	10	10	10	10	10	10
22 → 29	Mean	16	15.58	15.5	15.76
	SD	0.24	0.88	0.66	0.93
	N	10	10	10	10
29 → 36	Mean	16.4	16.36	16.93	16.68
	SD	0.47	1.56	1.09	0.87
	N	10	10	10	10
36 → 43	Mean	15.13	15.25	15.91	15.2
	SD	0.57	1.64	1.05	0.89
	N	10	10	10	10

ANOVA and Dunnett test (Log)

* p < 0.05

Table 4: Summary of oestrous cycle length prior to cohabitation period

Day(s) Relative to Start		Date	Females
			0	100	300	1000
			mg/kg bw/day
Mean	1 → 15 [a]	Mean	4	4.3	4.2	4.6
Cycle days)		SD	0	0.3	0.3	0.9
		N	10	10	10	10

ANOVA and Dunnett test (Log)

Table 5: Summary of maternal body weights and body weight gain during gestation and lactation period

Day(s) Relative to Mating			Females				Day(s) Relative to Littering		Females
			0	100	300	1000			0	100	300	1000
			mg/kg bw/day						mg/kg bw/day
Body weight (g)	0 [a]	Mean	202.26	205.16	204.37	208.04	0 [a]	Mean	217.16	214.1	222.79	224.03
		SD	11.05	8.67	16.37	12.96		SD	15.27	10.09	13.48	22.52
		N	10	9	10	10		N	9	9	10	10
	7 [a]	Mean	221.18	224.15	224.67	230.75	4 [a]	Mean	223.05	225.2	233.29	231.78
		SD	10.23	11.14	15.22	14.44		SD	15.61	9.61	14.17	20.16
		N	10	9	10	10		N	9	9	9	10
	14 [a]	Mean	246.02	248.35	249.87	256.83	7 [a]	Mean	231.92	232.98	235.73	240.83
		SD	11.83	11.15	16.15	19.27		SD	18.34	12.78	21.01	18.68
		N	10	9	10	10		N	9	9	9	9
	20 [a]	Mean	293.6	295.93	300.27	308.17	13 [a]	Mean	236.55	235.35	242.55	250.98
		SD	16.72	13.18	19.94	18.64		SD	18.62	13.05	14.53	23.01
		N	9	9	10	10		N	9	9	9	9
Absolute weight gain (g)	0 → 7 [a]	Mean	18.92	18.98	20.3	22.71	0 → 4 [a]	Mean	5.89	11.1	11.3	7.75
		SD	4.24	3.76	3.3	4.1		SD	6.13	6.66	5.42	7.22
		N	10	9	10	10		N	9	9	9	10
	7 → 14 [a]	Mean	24.84	24.2	25.2	26.07	4 → 7 [a]	Mean	8.87	7.77	2.44	6.84
		SD	3.16	2.87	3.08	6.35		SD	5.23	8.87	8.69	7.93
		N	10	9	10	10		N	9	9	9	9
	14 → 20 [a]	Mean	48.18	47.58	50.41	51.34	7 → 13 [a]	Mean	4.63	2.37	6.82	10.15
		SD	5.42	3.46	6.84	5.89		SD	8.88	5.78	9.07	10.28
		N	9	9	10	10		N	9	9	9	9
	0 → 20 [a]	Mean	92.44	90.76	95.9	100.13	0 → 13 [a]	Mean	19.39	21.25	20.56	23.58
		SD	9.34	5.75	6.75	10.71		SD	8.31	10.21	6.7	9.99
		N	9	9	10	10		N	9	9	9	9

[a] ANOVA and Dunnett test (Log)

Table 6: Summary of maternal food consumption during gestation and lactation period

Day(s) Relative to Mating			Females				Day(s) Relative to Littering		Females
			0	100	300	1000			0	100	300	1000
			mg/kg bw/day						mg/kg bw/day
Total Food Consumption (g)	0 → 7 [a]	Mean	97.97	103.17	99.19	106.3	0 → 4 [a]	Mean	97.03	96.9	103.69	93.8
		SD	6.79	9.01	9.92	9.84		SD	26.92	8.83	18.58	18.75
		N	10	9	10	10		N	9	9	9	10
	7 → 14 [a]	Mean	121.13	123.03	123.02	130.9	4 → 13 [a]	Mean	337.72	337.35	335.7	343.46
		SD	10.05	10.41	8.5	17.7		SD	39.91	34.21	35.43	36.59
		N	10	9	10	10		N	9	9	9	9
	14 → 20 [a]	Mean	111.17	107.38	113.12	119.65	0 → 13 [a]	Mean	434.76	434.25	439.38	439.86
		SD	9.7	9.2	10.72	10.13		SD	64.32	31.58	52.13	49.97
		N	9	9	10	10		N	9	9	9	9
	0 → 20 [a]	Mean	331.5	333.58	335.33	356.85
		SD	21.5	22.55	24.48	31.55
		N	9	9	10	10
Food Mean Consumption (g/day)	0 → 7 [a]	Mean	14	14.74	14.17	15.19	0 → 4 [a]	Mean	24.26	24.23	25.92	23.45
		SD	0.97	1.29	1.42	1.41		SD	6.73	2.21	4.64	4.69
		N	10	9	10	10		N	9	9	9	10
	7 → 14 [a]	Mean	17.3	17.58	17.57	18.7	4 → 13 [a]	Mean	37.52	37.48	37.3	38.16
		SD	1.44	1.49	1.21	2.53		SD	4.43	3.8	3.94	4.07
		N	10	9	10	10		N	9	9	9	9
	14 → 20 [a]	Mean	18.53	17.9	18.85	19.94	0 → 13 [a]	Mean	33.44	33.4	33.8	33.84
		SD	1.62	1.53	1.79	1.69		SD	4.95	2.43	4.01	3.84
		N	9	9	10	10		N	9	9	9	9
	0 → 20 [a]	Mean	16.57	16.68	16.77	17.84
		SD	1.08	1.13	1.22	1.58
		N	9	9	10	10

[a] ANOVA and Dunnett test (Log)

Table 7: Summary of mean pup body weights, ano-genital distance (AGD)m Ano-genital ratio (AGR) and ano-genital index (AGI)

Day(s) Relative to Littering			Females
			0	100	300	1000
			mg/kg bw/day
Litter Mean Pup BW - M	0 [a]	Mean	5.74	5.43	5.98	5.83
		SD	0.58	0.52	0.59	0.81
		N	9	9	9	10
	4 [a]	Mean	8.67	8.01	8.74	8.81
		SD	1.39	0.89	1.21	1.79
		N	9	9	9	9
	7 [a]	Mean	12.38	11.84	12.53	12.84
		SD	1.46	1.07	1.5	2.26
		N	9	9	9	9
	13 [a]	Mean	20.62	20.38	20.75	21.71
		SD	1.98	1.35	2.25	2.7
		N	9	9	9	9
Litter Mean Pup BW - F	0 [a]	Mean	5.68	5.25	5.68	5.63
		SD	0.57	0.5	0.5	0.78
		N	9	9	9	10
	4 [a]	Mean	8.55	7.86	8.28	8.76
		SD	1.09	1.04	0.96	1.81
		N	9	9	9	9
	7 [a]	Mean	12.26	11.43	11.99	12.59
		SD	1.41	1.31	1.34	2.06
		N	9	9	9	9
	13 [a]	Mean	20.47	19.7	20.01	21.4
		SD	1.74	0.92	2.04	2.7
		N	9	9	9	9
Litter Mean Pup BW	0 [a]	Mean	5.69	5.33	5.85	5.72
		SD	0.55	0.5	0.55	0.77
		N	9	9	9	10
	4 [a]	Mean	8.6	7.91	8.54	8.77
		SD	1.22	0.94	1.12	1.8
		N	9	9	9	9
	7 [a]	Mean	12.32	11.61	12.3	12.68
		SD	1.39	1.13	1.46	2.08
		N	9	9	9	9
	13 [a]	Mean	20.55	20.02	20.41	21.53
		SD	1.8	0.97	2.14	2.62
		N	9	9	9	9
Mean of pup AGD males	0 [a]	Mean	3.7	3.7	3.69	3.69
		SD	0.04	0.07	0.06	0.14
		N	9	9	9	10
Mean of pup AGD females	0 [a]	Mean	1.69	1.67	1.66	1.65
		SD	0.04	0.05	0.06	0.06
		N	9	9	9	10
Mean of pup AGR males	0 [a]	Mean	2.07	2.11	2.04	2.05
		SD	0.05	0.04	0.05	0.04
		N	9	9	9	10
Mean of pup AGR females	0 [a]	Mean	0.95	0.96	0.93	0.93
		SD	0.04	0.03	0.02	0.03
		N	9	9	9	10
Mean of pup AGI males	0 [a]	Mean	2.1	2.1	2	2.1
		SD	0.1	0	0	0
		N	9	9	9	10
Mean of pup AGI females	0 [a]	Mean	0.9	1	0.9	0.9
		SD	0	0	0	0
		N	9	9	9	10

[a] ANOVA and Dunnett test (Log)

Table 8: Summary of fertility data

Day(s) Relative to Mating			Males and females
			0	100	300	1000
			mg/kg bw/day
Paired Males	-	N	10	10	10	10
Paired Females	-	N	10	10	10	10
Mated	-	N+ve	10	10	10	10
Fertile Male	-	N+ve	10	9	10	10
Fertile Female	-	N+ve	10	9	10	10
M Mating Index	- [a]	%	100	100	100	100
F Mating Index	- [a]	%	100	100	100	100
Pregnant	-	N+ve	10	9	10	10
Littered	-	N+ve	9	9	10	10
Pre-coital Interval (f) (days)	- [a]	Mean SD N	2.2	2	2.1	2.3
			1.2	1.2	0.9	1.1
			10	10	10	10
F Fertility Index	- [a]	%	100	90	100	100
M Impregnated Female	- [a]	%	100	90	100	100

[a] ANOVA and Dunnett test (Log)

M- male

F- female

Table 9: Summary of litter data

Day(s) Relative to Littering			Females
			0	100	300	1000
			mg/kg bw/day
Littered	-	N+ve	9	9	10	10
Gestation Length (days)	- [a]	Mean	22.4	22.1	22.5	22.5
		SD	0.5	0.3	0.7	0.5
		N	9	9	10	10
Total No of Pups Born Day 0	-	Mean	11	12	10	11
		SD	2	2	4	2
		N	9	9	10	10
		Sum	99	108	104	111
Dead/Canibalism	LD 0	Sum	3	.	2	8
Live Male Pups	LD 0	Sum	41	53	53	48
Live Female Pups	LD 0	Sum	55	55	49	55
Live Pups Day 0	- [a]	Mean	10.7	12	11.3	10.3
		SD	1.3	2	2.2	2.1
		N	9	9	9	10
		Sum	96	108	102	103
Live Birth Index (%) Day 0	- [a1]	Mean	97.3	100	90	94.7
		SD	5.4	0	31.6	16.9
		N	9	9	10	10
Sex Ratio Day 0	- [a1]	Mean	42.5	49.2	56.7	44.1
Dead/Cannibalism	LD 1	Sum	1	1	.	2
Live Pups Day 1	-	Mean	10.6	11.9	11.3	10.1
		SD	1.1	1.9	2.2	2.5
		N	9	9	9	10
		Sum	95	107	102	101
Dead/Cannibalism	LD 2-4	Sum	4	1	.	12
Live Pups Day 4	- [a]	Mean	10.1	11.8	11.3	9.9
		SD	1.1	1.8	2.2	2.6
		N	9	9	9	9
		Sum	91	106	102	89
No. of Pups Culled D4	-	Sum	19	34	30	21
No.of Live Pup Post Cu	ll -	Sum	72	72	72	68
Survival Index on LD4	- [a]	Mean	95.6	98.4	100	86.2
		SD	10.3	3.1	0	31.6
		N	9	9	9	10
Dead/Cannibalism	LD 5-7	Sum	1	0	0	0
Live Pups Day 7	- [a1]	Mean	7.9	8	8	7.6
		SD	0.3	0	0	1
		N	9	9	9	9
		Sum	71	72	72	68
Survival Index on LD7	- [a]	Mean	98.6	100	100	100
		SD	4.2	0	0	0
		N	9	9	9	9
Dead/Cannibalism	LD 8-13	Sum	0	0	0	0
Live Pups Day 13	-	Mean	7.9	8	8	7.6
		SD	0.3	0	0	1
		N	9	9	9	9
		Sum	71	72	72	68
No. Of Implantation	- [a1]	Mean	12.6	13	12.4	12.9
		SD	1.3	1.7	2.9	1.7
		N	10	9	10	10
		Sum	126	117	124	129
Post-implantation Loss (%)	- [p]	Mean	14.62	7.86	22.56	18.61
		SD	10.85	7.92	27.75	19.33
		N	9	9	10	10

[a] - Anova & Dunnett(Arc SineSQRT)

[a1] - Anova & Dunnett(Log)

[p] - T-Test(Arc SineSQRT): [G1vsG2, G1vsG3, G1vsG4]

Applicant's summary and conclusion

Conclusions:

The test item had no effect on reproductive performance.