Ethylene di(acetate)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity to reproduction (OECD 421, Reproduction/Developmental Toxicity Screening Test), rat: NOAEL ≥ 1000 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

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

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

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

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

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

Dose / conc.:

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

Dose / conc.:

300 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:

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.

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

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: oestrous cycle:

no effects observed

Reproductive performance:

no effects observed

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.

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

Key result

Critical effects observed:

no

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Key result

Critical effects observed:

no

Reproductive effects observed:

no

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]

Conclusions:

The test item had no effect on reproductive performance.

Reference 2

Endpoint:

reproductive toxicity, other

Remarks:

male fertility

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Only few details given in a publication.

Principles of method if other than guideline:

Effects of ethylene glycol diacetate by oral gavage on testis of mice.

GLP compliance:

no

Species:

mouse

Strain:

other: JCL-ICR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CLEA Co., Japan
- Age at study initiation: 6 weeks

Route of administration:

oral: gavage

Vehicle:

other: water or olive oil (not further specified)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

(P) Males: 35 days

Frequency of treatment:

daily, 5 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

BODY WEIGHT: Yes (No further information)

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All animals were sacrificed on the following day of the final administration.

GROSS NECROPSY
- The animals were necropsied under pentobarbital sodium anesthesia.

HISTOPATHOLOGY / ORGAN WEIGHTS
Tissues specimens for histopathological examination were fixed in 10% buffered formalin solution, embedded in paraffin, sectioned and stained with hematoxylin and eosin. The testes were weighed and the combined weight of seminal vesicles and coagulating gland was measured.

HAEMATOLOGY
Blood was taken from the posterior vena cava at the time of necropsy.

Clinical signs:

not examined

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

ORGAN WEIGHTS (PARENTAL ANIMALS)
No effects on testis weights were observed in the test animals.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No effects reported in mice treated with ethylene diacetate.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No effects reported in mice treated with ethylene diacetate.

HAEMATOLOGY (PARENTAL ANIMALS)
No effects on white blood cell count, red blood cell count, packed cell volume and/or haemoglobin content in the animals treated with ethylene diacetate.

Dose descriptor:

other: effects on testis

Effect level:

>= 2 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Critical effects observed:

not specified

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable (Klimisch score 1) study performed with the registered substance, and is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.6, of Regulation (EC) No. 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Toxicity to reproduction, oral route:

One oral gavage reproduction/developmental toxicity screening test and a publication are available investigating toxic effects on reproduction induced by ethylene diacetate (CAS 111 -55 -7). The publication by Nagano (1984) provides only limited data investigating the effect on testis of mice after repeated oral administration via gavage. The test item was administered to male mice for 35 days, 5 days/week at concentrations of 500, 1000 and 2000 mg/kg bw/day. In the study, no effects on testis weights, gross pathology and histopathological parameters were reported. In addition, no effects on white blood cell count, red blood cell count, packed cell volume and/or haemoglobin content were observed.

In addition, an oral gavage reproduction/developmental toxicity screening test was performed according to OECD guideline 421 and in compliance with GLP (Eurofins, 2018c). Male and female rats were exposed to the test item at doses of 100, 300 and 1000 mg/kg bw/day. The test substance was dissolved in corn oil and orally administered to 10 animals per sex per day. A similar constituted group received the vehicle and served as control. Males were treated for a period of 42 days, whereas females were exposed to the test substance for a maximum of 56 days starting 14 days prior to mating and until lactation day 13. Clinical sings of toxicity were not observed and no treatment-related mortality was recorded. Body weights were not adversely affected by treatment. Body weight gains in female rats treated at 1000 mg/kg bw/day were significantly higher compared to other dose groups, which was considered toxicologically not significant as there were no significant differences observed in weekly main body weights and food consumption. Food consumption was not altered throughout the treatment period when compared to vehicle control. An incidence of significant increase in the food consumption of female rats during days 1-8 in the 100 mg/kg bw/day dose group was not considered treatment-related as there was no dose-response relationship.In parental animals, no effects on reproductive function (including gestation length, oestrus cycle and spermatogenesis) and performance (including mating, fertility, pre-coital intervals and implantation) were observed after treatment compared to controls. Gross necropsy and histopathology of preserved organs (including 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 and the interstitial testicular cell structure) did not reveal any substance-related effects in the parental animals. In addition, no toxicologically relevant alterations in offspring viability indices were observed. Based on these results and under the conditions of this study, a No Observed Adverse Effect Level (NOAEL) for reproductivity of ≥ 1000 mg test item/kg bw/day was derived for Wistar rats.

Effects on developmental toxicity

Description of key information

Developmental toxicity (OECD 414 and 421), rat: NOAEL ≥ 1000 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: In vitro data; no approved test system

Principles of method if other than guideline:

The hazard potential of a test substance to developmental biology is assessed by an in vitro short-term screening test in fresh water coelenterate Hydra attenuata. The "hydra assay" is evaluated determining the difference between the lowest dose or concentration overtly toxic to adults (A) and the lowest concentration interfering with development (D) of an artificial "embryo" of reaggregated adult hydra cells and the A/D ratio.

GLP compliance:

not specified

Species:

other: in vitro test

Strain:

other: Hydra attenuata

Details on test animals or test system and environmental conditions:

Adult polyps of the fresh water coelenterate Hydra attenuata were grown in a specially prepared apparatus where they would reproduce by asexual budding and could be fed and cleaned of expelled detritus on a regular schedule with a minimum of technical effort.
Experimentation established that randomly reaggregated cells undergo a full ontogenesis sequence and give rise to a new population of adult polyps in about 92 h if they are left undisturbed. These artificial preparations consist of two broad classes of cells: fully differentiated adult cells which quickly achieve spatial orientation and a much smaller number of undifferentiated interstitial cells capable to becoming any of the numerous differentiated cells typical of adult hydra. During their 92 h period of differentiation and organogenesis, these artificially manufactured pellets achieve all of the developmental biologic phenomena known to occur in an embryo of any species and are thus termed artificial "embryos".

Route of administration:

other: not applicable (in vitro test)

Type of inhalation exposure (if applicable):

other: not applicable (in vitro test)

Vehicle:

water

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

not applicable (in vitro test)

Duration of treatment / exposure:

92 h

Frequency of treatment:

not applicable (in vitro test)

Duration of test:

92 h

Dose / conc.:

0.001 other: mL/L

Remarks:

step 1, Basis: nominal conc.

Dose / conc.:

0.01 other: mL/L

Remarks:

step 1, Basis: nominal conc.

Dose / conc.:

0.1 other: mL/L

Remarks:

step 1, Basis: nominal conc.

Dose / conc.:

1 other: mL/L

Remarks:

step 1, Basis: nominal conc.

Dose / conc.:

10 other: mL/L

Remarks:

step 1, Basis: nominal conc.

Dose / conc.:

100 other: mL/L

Remarks:

step 1, Basis: nominal conc.

Dose / conc.:

1 000 other: mL/L

Remarks:

step 1, Basis: nominal conc.

Dose / conc.:

0.1 other: mL/L

Remarks:

step 2, Basis: nominal conc.

Dose / conc.:

0.2 other: mL/L

Remarks:

step 2, Basis: nominal conc.

Dose / conc.:

0.3 other: mL/L

Remarks:

step 2, Basis: nominal conc.

Dose / conc.:

0.4 other: mL/L

Remarks:

step 2, Basis: nominal conc.

Dose / conc.:

0.5 other: mL/L

Remarks:

step 2, Basis: nominal conc.

Dose / conc.:

0.6 other: mL/L

Remarks:

step 2, Basis: nominal conc.

Dose / conc.:

0.7 other: mL/L

Remarks:

step 2, Basis: nominal conc.

Dose / conc.:

0.8 other: mL/L

Remarks:

step 2, Basis: nominal conc.

Dose / conc.:

0.9 other: mL/L

Remarks:

step 2, Basis: nominal conc.

Dose / conc.:

1 other: mL/L

Remarks:

step 2, Basis: nominal conc.

No. of animals per sex per dose:

other: 700 - 1000 adult hydra per assay

Control animals:

other: not applicable (in vitro test)

Details on study design:

At the beginning of each assay, approx. 700 - 1000 clean adult hydra were collected and then dissociated mechanically into their component cells which were then randomly reassociated by being gently packed into small pellets through low-speed centrifugation.

The protocol was a three-step procedure that was used twice; once for embryos and once for adult hydra.
- Step 1: test substance concentrations from 1E-03 to 1E03 mL/L to identify the lowest concentration of the chemical capable of disrupting development of the embryo and producing signs of toxicity in adults, respectively.
- Step 2: The second step of the testing protocol again investigates the lowest toxic whole-log concentration eliciting the toxic endpoints obtained in the first step as well as the next lowest whole-log concentration, i.e., the highest whole-log no-effect level. The main part of step 2 is to test the eight 1/10 log concentrations between these two whole-log concentrations. This step determines to within 1/10 log those minimal effective concentrations (MEC) of the test substance capable of producing adult and developmental toxicity.
- Step 3: confirmation of the MEC observed in step 2. The adult MEC (A) and the developmental MEC (D) are calculated as a ratio. This A/D ratio is considered as a developmental toxicity hazard index whose increasing size is directly proportional to a chemical´s ability to injure embryos in the absence of adult toxicity.

Dose descriptor:

other: adult MEC (A); "embryo" MEC (D)

Effect level:

1

Based on:

test mat.

Basis for effect level:

other: other:

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:not examined

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

The result of the in vitro hydra screening assay is, that the adult and the "embryo" were injured at the same concentration. Thus, no specific developmental toxicity was observed. This test system can predict the A/D ratio but is unsuitable for quantitative risk estimation because the doses in hydra are often different from those needed to produce mammalian toxicity. As the test system is not accepted, the data is not sufficient for assessment of developmental toxicity.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Apr - 11 Sep 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22 Jan 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Limit test:

no

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

Species:

rat

Strain:

Wistar

Remarks:

Han_Tac: WH

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: 13 - 14 weeks
- Weight at study initiation: 186.55 to 260.87 g
- Housing: in sterilised standard polysulfone rat 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
Pre-mating: two rats of the same sex per cage
Mating: female rats were cohabited with males in a 1:1 ratio in the same cage
Post-mating/Treatment: After mating confirmation, females were housed individually

- 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 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared at 4 days intervals. The required quantities were weighed and a small volume of vehicle (corn oil) was added and mixed well using a glass rod. The final volume was made up with the vehicle to receive the required final concentration. The volume was made up to the upper meniscus during dose formulation.

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: 25, 75 and 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations were analysed for the test item concentration at the initiation of treatment and at termination of treatment period (1-3 days before termination). 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, if the overall mean result of all layers was within ± 15.0% of the claimed concentration and the relative standard deviation was equal or less than 10.0%. The results of dose formulations were within the acceptable limits for all dose groups.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy

Duration of treatment / exposure:

Day 5 - 19 of gestation

Frequency of treatment:

daily, 7 days/week

Duration of test:

Day 20 of gestation

Dose / conc.:

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

Dose / conc.:

300 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:

24 parental females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on a preliminary range-finding study, in which animals were orally exposed to up to 1000 mg/kg bw/day for 14 days (Study N3725). The results indicated that treatment did not cause any adverse effects up to the highest dose of 1000 mg/kg bw/day during the 14 day treatment period. Therefore, 100, 300 and 1000 mg/kg bw/day were selected as the dose levels for the main study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 3, 5, 8, 11, 14, 17 and 20

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION : No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: gravid uterus, cervix

Ovaries and uterine content:

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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter

Statistics:

- Levene’s test for homogeneity of intra group variances
- Analysis of Variance (ANOVA) was performed after suitable transformation of data on maternal body weight, body weight in interval, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption
- Dunnett’s pairwise comparison of the treated group means with the control group (in case of statistical significance)
- Analysis of Covariance (ANCOVA) for analysis of fetal weight for male and female taking litter size as covariante for group
- Kruskal wallis test for group comparison regarding the number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss, external, visceral and skeletal observations for variations
- Cochran Armitage trend test followed by Fisher’s exact test for group association testing the incidence of with and without resorptions in dams

Indices:

Maternal parameters:
- Mean number of corpora lutea/group = Total number of corporar lutea/Total number of pregnant animals
- Mean number of implantations/group = Total number of implantations/Total number of pregnant animals
- Embryonic resorption index [%] = Number of early resorptions/Number of implantations x 100
- Fetal resorption index [%] = Number of late resorptions/Number of implantations x 100
- Pre-implantation loss per group [%] = (Number of CL – Number of implantations)/Number of CL x 100
- Post-implantation loss per group [%] =n Number of (early + late) resorptions/ Total number of implantations x 100
- Implantation index [%] = Number of implantations sites/Number of corpora lutea x 100

Litter data:
- Mean litter size per group = Total number of fetuses/Total number of pregnant animals
- Percentage of abnormal fetuses = Total abnormal fetuses/Total number of fetuses x 100
- Percentage of live fetuses per group (Live fetus index) = Number of live fetuses/Total number of fetuses x 100
- Percentage of dead fetuses per group (Dead fetus index) = Number of dead fetuses/Total number of fetuses x 100
- Percentage of male fetuses per group = Number of male fetuses/Total number of fetuses x 100
- Sex ratio (F:M) = Number of females/Number of males
- Corrected body weight (Carcass weight) = Terminal body weight (body weight gain on GD20) – unopened uterine weight
- Corrected body weight gain = Corrected body weight – body weight on GD5

Historical control data:

Historical data was provided to allow comparison with concurrent controls. The following data was given:
- Mean maternal body weight during gestation
- Body weight gain during gestation
- Food intake
- Maternal data
- Litter data
- External observations
- Visceral observations
- Sekeletal observations

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no effects observed

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: one small fetus

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: increase in percental fetal incidence of delayed skeletal ossification

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: single incidence of moderate renal pelvis dilation of kidney

Details on embryotoxic / teratogenic effects:

EXTERNAL MALFORMATIONS:
There was an incidence of a small fetus in the high dose group. This finding was not of any toxicological significance as this is commonly observed in rat fetuses.

SKELETAL MALFORMATIONS:
There was a statistically significant increase in the percent fetal incidence of delayed skeletal ossification in one of the bone component (Forelimb metacarpal 1/4) observed in the 1000 mg/kg bw/day dose group as compared to the vehicle control group. The effect was considered not significant because historical data shows that this normal variation was commonly observed in rat fetuses.
Furthermore, there was a statistically significant increase in the percent fetal incidence of asymmetrical ossification of sternum numbers 4 and 5 in the high dose group (1000 mg/kg bw/day). As maternal and litter parameters at this dose were not affected, the finding was not considered as an adverse effect.

VISCERAL MALFORMATIONS:
There was a single incidence of moderate renal pelvis dilation of kidney in the high dose group. This finding was comparable to historical data and hence is not of any significance.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1: Summary of clinical signs and mortality:

	Group No.	G1	G2	G3	G4
Observations	Dose (mg/kg/day)	0	100	300	1000
Total No. of rats found sperm positive		24	24	24	24
Clinical signs		NAD	NAD	NAD	NAD
Mortality		-------------None------------

NAD: no abnormality detected

Table 2: Summary on maternal group body weight

Group	Dose	No. Of	Group mean body weight (g) on gestation day (GD)
No.	(mg/kg/day)	Rats$		0	3	5	8	11	14	17	20
G1	0	23	Mean	218.5	227.63	233.22	240.46	253.65	264.62	289.32	314.74
			SD	14.83	14.47	15.34	16.65	17.38	18.26	21.07	27.76
G2	100	24	Mean	221.82	230.99	236.15	243.92	257.93	268.5	293.56	314.59
			SD	14.7	14.43	14.09	15.6	16.95	18.11	20.71	21.58
G3	300	23	Mean	219.76	229	234.99	242.64	257.12	268.53	291.09	311.35
			SD	14.47	14.59	14.02	14.71	16.43	17.66	21.48	24.43
G4	1000	22	Mean	222.73	231.41	236.7	242.8	255.86	266.43	288.86	309.12
			SD	16.21	16.84	17.08	17.13	17.55	19.06	21.08	21.81

$: Pregnant rats

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

Period of treatment (days of gestation)	Group No.	G1	G2	G3	G4
	Dose (mg/kg/day)	0	100	300	1000
	No. of Rats$	23	24	23	22
Intermittent food intake
0-3	Mean	15.63	16.13	16.33	15.61
	SD	1.74	1.44	1.28	1.61
3-5	Mean	17.40	17.78	17.87	17.56
	SD	2.30	1.33	1.47	2.11
5-8	Mean	16.21	16.66	16.95	15.99
	SD	2.38	2.15	1.47	1.74
8-11	Mean	17.30	17.84	18.05	17.17
	SD	2.44	1.55	1.54	1.60
11-14	Mean	18.04	18.74	19.14	18.36
	SD	2.34	1.53	1.26	2.07
14-17	Mean	19.08	19.36	19.54	18.87
	SD	2.61	1.93	2.14	2.06
17-20	Mean	16.51	16.78	17.34	16.91
	SD	3.37	2.70	3.10	4.11

$: Pregnant rats

Table 4: Summary of maternal data

Parameters	Group No.	G1	G2	G3	G4
	Dose (mg/kg/day)	0	100	300	1000
	No. of Rats$	23	24	23	22
Gravid uterine weight (g)	Mean	64.47	68.22	61.54	58.91
	SD	13.38	12.24	17.11	12.54
Number of Corpora lutea	Mean	13.22	14	13.52	14
	SD	1.95	2.43	1.86	1.8
Number of Implantations	Mean	11.74	12.79	11.87	11.59
	SD	2.47	2.6	2.91	2.26
Early Resorptions	Mean	0.3	0.46	0.78	0.82
	SD	0.56	0.72	1.28	1.22
Late Resorptions	Mean	0.04	0	0.04	0.14
	SD	0.21	0	0.21	0.35
Pre-implantation Loss	Mean	1.48	1.21	1.65	2.41
	SD	1.65	1.35	1.56	2.17
Post-implantation Loss	Mean	0.35	0.46	0.83	0.95
	SD	0.65	0.72	1.3	1.17
Dams with any Resorption	Total	6	9	9	12
Early Resorptions (%)	Mean	2.62	3.52	7.19	7.83
	SD	4.63	5.21	12.15	12.55
Late Resorptions (%)	Mean	0.33	0	0.31	1.21
	SD	1.6	0	1.49	3.14
Pre-implantation Loss (%)	Mean	11.27	8.95	13.69	16.82
	SD	12.82	10.62	17.76	13.66
Post-implantation Loss (%)	Mean	2.95	3.52	7.5	9.04
	SD	5.25	5.21	12.24	12.15
Implantation Index (%)	Mean	88.73	91.05	86.31	83.18
	SD	12.82	10.62	17.76	13.66

$: Pregnant rats

Table 5: Summary of litter data

Parameters	Group No.	G1	G2	G3	G4
	Dose (mg/kg/day)	0	100	300	1000
	No. of Rats$	23	24	23	22
No. of litters		23	24	23	22
Total no. of fetuses		262	296	254	234
Mean litter size		11.4	12.3	11	10.6
Total live fetuses
a. Number		262	296	254	234
b.  Weight (g)	Mean	3.76	3.8	3.73	3.61
	SD	0.47	0.2	0.22	0.27
Live male fetuses
a. Number		136	163	136	119
b.  Weight (g)	Mean	3.85	3.88	3.78	3.73
	SD	0.45	0.23	0.22	0.26
Live female fetuses
a. Number		126	133	118	115
b.  Weight (g)	Mean	3.65	3.7	3.7	3.55
	SD	0.54	0.2	0.29	0.32
Sex Ratio - Male : Female		1:0.93	1:0.82	1:0.87	1:0.97
(% of male fetuses)		-51.90%	-55.10%	-53.50%	-50.90%

$: Pregnant rats

Table 6: Summary of gross pathological findings

Group No.	G1	G2	G3	G4
Parameters Dose (mg/kg/day)	0	100	300	1000
1.  No. of rats subjected to caesarean section	24	24	24	24
2.  No. of rats pregnant at caesarean section	23	24	23	22
3.  No. of rats showing gross pathology	0	0	0	0

Table 7: Summary of fetal external observations (incidence and percentage)

Group No.	G1				G2				G3				G4
Dose (mg/kg/day)	0				100				300				1000
No. of litters examined	23				24				23				22
No. of fetuses examined	262				296				254				234
	Fetus		Litter		Fetus		Litter		Fetus		Litter		Fetus		Litter
	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%
Normal Variant	None
Minor Anomalies
Small fetus	0	0	0	0	0	0	0	0	0	0	0	0	1	0.43	1	4.55
Major Malformations	None

Table 8: Summary of fetal visceral observations (incidence and percentage)

Group No.	G1				G2				G3				G4
Dose (mg/kg/day)	0				100				300				1000
No. of litters examined	23				24				23				22
No. of fetuses examined	127				140				120				112
	Fetus		Litter		Fetus		Litter		Fetus		Litter		Fetus		Litter
	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%
Normal Variant
Kidney-renal pelvis dilation	1	0.79	1	4.35	0	0	0	0	0	0	0	0	0	0	0	0
(slight)
Minor Anomalies
Kidney-renal pelvis dilation	0	0	0	0	0	0	0	0	0	0	0	0	1	0.89	1	4.55
(moderate)
Major Malformations	None

Table 9: Summary of fetal skeletal observations (incidencde and percentage)

Group No.	G1				G2				G3				G4
Dose (mg/kg/day)	0				100				300				1000
No. of litters examined	23				24				23				22
No. of fetuses examined	127				140				120				112
	Fetus		Litter		Fetus		Litter		Fetus		Litter		Fetus		Litter
Variant	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%
DSO:Ileum	1	0.74	1	4.35	0	0	0	0	0	0	0	0	0	0	0	0
DSO:Ischium,Pubis	1	0.74	1	4.35	0	0	0	0	0	0	0	0	0	0	0	0
DSO:Stern:# 2	0	0	0	0	0	0	0	0	0	0	0	0	1	0.82	1	4.55
DSO:Stern:# 3	0	0	0	0	0	0	0	0	0	0	0	0	1	0.82	1	4.55
DSO:Stern:# 5	2	1.48	2	8.7	10	6.41	8	33.33	6	4.48	4	17.39	6	4.92	3	13.64
DSO:Stern:# 6	3	2.22	3	13.04	5	3.21	5	20.83	4	2.99	2	8.7	6	4.92	3	13.64
DSO:Stern:# 1-6	1	0.74	1	4.35	0	0	0	0	0	0	0	0	2	1.64	2	9.09
DSO:Stern:# 4-6	1	0.74	1	4.35	0	0	0	0	0	0	0	0	0	0	0	0
DSO:Stern:# 5,6	2	1.48	2	8.7	1	0.64	1	4.17	2	1.49	2	8.7	8	6.56	4	18.18
DSO:Stern:# 2,4-6	0	0	0	0	0	0	0	0	0	0	0	0	1	0.82	1	4.55
DSO:CV:centra:1/7	0	0	0	0	0	0	0	0	0	0	0	0	1	0.82	1	4.55
DSO:CV:centra:2/7	1	0.74	1	4.35	0	0	0	0	1	0.75	1	4.35	3	2.46	3	13.64
DSO:CV:centra:3/7	3	2.22	1	4.35	2	1.28	2	8.33	4	2.99	4	17.39	13	10.66	11	50
DSO:CV:centra:4/7	9	6.67	5	21.74	8	5.13	6	25	13	9.7	10	43.48	14	11.48	12	54.55
DSO:CV:centra:5/7	3	2.22	3	13.04	7	4.49	5	20.83	8	5.97	6	26.09	8	6.56	6	27.27
DSO:CV:centra:6/7	8	5.93	5	21.74	10	6.41	5	20.83	17	12.69	8	34.78	11	9.02	7	31.82
DSO:CV:centra:7/7	3	2.22	2	8.7	2	1.28	2	8.33	1	0.75	1	4.35	7	5.74	6	27.27
DSO:CdV:centra:1/4	9	6.67	6	26.09	8	5.13	4	16.67	16	11.94	8	34.78	18	14.75	10	45.45
DSO:CdV:centra:2/4	1	0.74	1	4.35	0	0	0	0	0	0	0	0	2	1.64	2	9.09
DSO:CdV:centra:3/4	0	0	0	0	0	0	0	0	1	0.75	1	4.35	1	0.82	1	4.55
DSO:CdV:centra:4/4	3	2.22	1	4.35	0	0	0	0	0	0	0	0	3	2.46	3	13.64
DSO:CdV:arch:1/2	6	4.44	5	21.74	2	1.28	1	4.17	15	11.19	10	43.48	19	15.57	6	27.27
DSO:CdV:arch:2/2	3	2.22	1	4.35	0	0	0	0	0	0	0	0	4	3.28	4	18.18
DSO:TV:centra:1-13	1	0.74	1	4.35	0	0	0	0	0	0	0	0	0	0	0	0
DSO:LV:centra:6/6	1	0.74	1	4.35	0	0	0	0	0	0	0	0	0	0	0	0
DSO:SV:centra:4/4	1	0.74	1	4.35	0	0	0	0	0	0	0	0	0	0	0	0
DSO:SV:arch:4/4	1	0.74	1	4.35	0	0	0	0	0	0	0	0	0	0	0	0
DSO:F limb:Metacarp.:1/4	8	5.93	5	21.74	4	2.56	3	12.5	25	18.66	13	56.52	35	28.69*	14	63.64
DSO:F limb:Metacarp.:3/4	1	0.74	1	4.35	0	0	0	0	0	0	0	0	0	0	0	0
DSO:F limb:Pr. Phal:1/2	3	2.22	3	13.04	2	1.28	2	8.33	4	2.99	4	17.39	2	1.64	1	4.55
DSO:F limb:Pr. Phal:2/2	43	31.85	14	60.87	42	26.92	13	54.17	46	34.33	17	73.91	66	54.1	19	86.36
DSO:F limb:Dt. Phal:1/4	0	0	0	0	0	0	0	0	3	2.24	2	8.7	26	21.31	11	50
DSO:F limb:Dt. Phal:2/4	0	0	0	0	0	0	0	0	2	1.49	2	8.7	2	1.64	2	9.09
DSO:F limb:Dt. Phal:4/4	5	3.7	2	8.7	0	0	0	0	2	1.49	2	8.7	9	7.38	6	27.27
DSO:H limb:Metatar.:1/4	1	0.74	1	4.35	0	0	0	0	0	0	0	0	3	2.46	3	13.64
DSO:H limb:Metatar.:3/4	1	0.74	1	4.35	0	0	0	0	0	0	0	0	0	0	0	0
DSO:H limb:Dt. Phal:1/5	4	2.96	3	13.04	9	5.77	4	16.67	18	13.43	9	39.13	26	21.31	11	50
DSO:H limb:Dt. Phal:5/5	6	4.44	3	13.04	0	0	0	0	3	2.24	3	13.04	16	13.11	8	36.36
INO/PO:Skull bones	1	0.74	1	4.35	0	0	0	0	0	0	0	0	0	0	0	0
INO/PO:Parietal	0	0	0	0	0	0	0	0	0	0	0	0	1	0.82	1	4.55
INO/PO:Pa, Ipa	3	2.22	3	13.04	0	0	0	0	2	1.49	2	8.7	3	2.46	3	13.64
INO/PO:Inter pariet.	0	0	0	0	0	0	0	0	0	0	0	0	2	1.64	1	4.55
INO/PO:Supraocci	0	0	0	0	0	0	0	0	0	0	0	0	1	0.82	1	4.55
INO/PO:Pubis	0	0	0	0	0	0	0	0	0	0	0	0	1	0.82	1	4.55
INO/PO:Fr, Pa, Ipa	0	0	0	0	0	0	0	0	0	0	0	0	1	0.82	1	4.55
INO/PO:Stern:# 1	4	2.96	2	8.7	0	0	0	0	0	0	0	0	3	2.46	3	13.64
INO/PO:Stern:# 2	7	5.19	6	26.09	5	3.21	5	20.83	5	3.73	4	17.39	15	12.3	8	36.36
INO/PO:Stern:# 3	4	2.96	2	8.7	0	0	0	0	0	0	0	0	1	0.82	1	4.55
INO/PO:Stern:# 4	3	2.22	3	13.04	1	0.64	1	4.17	2	1.49	2	8.7	6	4.92	5	22.73
INO/PO:Stern:# 5	41	30.37	17	73.91	51	32.69	21	87.5	47	35.07	20	86.96	36	29.51	16	72.73
INO/PO:Stern:# 6	13	9.63	10	43.48	16	10.26	11	45.83	19	14.18	10	43.48	20	16.39	13	59.09
INO/PO:CV:centra:1/7	0	0	0	0	0	0	0	0	3	2.24	3	13.04	1	0.82	1	4.55
INO/PO:CV:centra:2/7	1	0.74	1	4.35	0	0	0	0	4	2.99	4	17.39	2	1.64	2	9.09
INO/PO:CV:centra:3/7	1	0.74	1	4.35	1	0.64	1	4.17	1	0.75	1	4.35	1	0.82	1	4.55
INO/PO:CV:centra:4/7	0	0	0	0	1	0.64	1	4.17	0	0	0	0	1	0.82	1	4.55
INO/PO:TV:centra:1/13	0	0	0	0	0	0	0	0	0	0	0	0	1	0.82	1	4.55
HYPOPLASTIC:Stern:# 1	0	0	0	0	0	0	0	0	0	0	0	0	1	0.82	1	4.55
HYPOPLASTIC:Stern:# 2	2	1.48	1	4.35	1	0.64	1	4.17	0	0	0	0	1	0.82	1	4.55
HYPOPLASTIC:Stern:# 3	0	0	0	0	0	0	0	0	0	0	0	0	1	0.82	1	4.55
HYPOPLASTIC:Stern:# 5	10	7.41	6	26.09	13	8.33	9	37.5	7	5.22	6	26.09	4	3.28	3	13.64
SPLIT:TV:centra:1/13	0	0	0	0	0	0	0	0	0	0	0	0	1	0.82	1	4.55
DB:TV:centra:1/13	4	2.96	3	13.04	2	1.28	2	8.33	6	4.48	5	21.74	10	8.2	8	36.36
DB:TV:centra:2/13	1	0.74	1	4.35	1	0.64	1	4.17	1	0.75	1	4.35	1	0.82	1	4.55
ASY DB:TV:centra:1/13	1	0.74	1	4.35	4	2.56	4	16.67	1	0.75	1	4.35	1	0.82	1	4.55
ASY OSSI:stern:# 3,4	0	0	0	0	3	1.92	3	12.5	1	0.75	1	4.35	2	1.64	2	9.09
ASY OSSI:stern:# 4	0	0	0	0	0	0	0	0	2	1.49	2	8.7	2	1.64	2	9.09
ASY OSSI:stern:#2-4	0	0	0	0	0	0	0	0	0	0	0	0	1	0.82	1	4.55
ASY OSSI:stern:#4,5	0	0	0	0	0	0	0	0	1	0.75	1	4.35	5	4.10*	5	22.73
RUDIMENTARY: RIB(Rt/Lt/B):# 14	31	22.96	17	73.91	35	22.44	16	66.67	35	26.12	17	73.91	35	28.69	15	68.18
ACCESSORY: RIB(Rt/Lt/B):# 14	1	0.74	1	4.35	2	1.28	2	8.33	1	0.75	1	4.35	3	2.46	3	13.64
WAVY:RIB(Rt/Lt/B)(+/++):1/13	1	0.74	1	4.35	0	0	0	0	0	0	0	0	0	0	0	0
WAVY:RIB(Rt/Lt/B)(+/++):2/13	2	1.48	2	8.7	0	0	0	0	1	0.75	1	4.35	0	0	0	0
WAVY:RIB(Rt/Lt/B)(+/++):3/13	1	0.74	1	4.35	0	0	0	0	0	0	0	0	0	0	0	0
WAVY:RIB(Rt/Lt/B)(+/++):5/13	0	0	0	0	0	0	0	0	1	0.75	1	4.35	0	0	0	0
EXTRA:RIB(Rt/Lt/B):# 14	0	0	0	0	0	0	0	0	0	0	0	0	3	2.46	2	9.09
EXTRA:LV:centra & arch #7	0	0	0	0	0	0	0	0	0	0	0	0	3	2.46	3	13.64

Conclusions:

The test item had no effect on intrauterine development.

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 available information comprises an adequate and reliable (Klimisch score 1) study performed with the registered substance, and is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.6, of Regulation (EC) No. 1907/2006.

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

Data on developmental toxicity induced by ethylene diacetate (CAS 111 -55 -7) comprises one publication on in vitro developmental toxicity screening, and two experimental studies, one reproduction/developmental toxicity screening test performed according to OECD guideline 421 and a prenatal developmental toxicity study according to OECD guideline 414.

A publication investigating the developmental toxicity of ethylene diacetate (CAS 111 -55 -7) in vitro is available (Johnson, 1984). The hazard potential of a test substance to developmental biology is assessed by an in vitro short-term screening test in fresh water coelenterate Hydra attenuata. The "hydra assay" is evaluated determining the difference between the lowest dose or concentration overtly toxic to adults (A) and the lowest concentration interfering with development (D) of an artificial "embryo" of re-aggregated adult hydra cells and the A/D ratio. The result of the present in vitro hydra screening assay described in this publication is, that the adult and the "embryo" were injured at the same concentration. The authors concluded that therefore no specific developmental toxicity was observed. However, as the test system is not accepted, the data is not sufficient for assessment of developmental toxicity.

 

Second, an oral gavage reproduction/developmental toxicity screening test was performed according to OECD guideline 421 and in compliance with GLP (Eurofins, 2018c). The study has been discussed in detail above (toxicity to reproduction). Here, only findings related to developmental toxicity are discussed.

No treatment-related changes were observed in litter parameters comprising of total number of fetuses, number and weight of male and female fetuses and sex ratio at all dose groups. Mortality rates in the litter were comparable to those of control animals. In addition, there were no treatment-related clinical signs of toxicity or external abnormalities in the parents or the litters. The mean litter sizes and number of dead pups at first observation were comparable to vehicle control treated animals and survival data of pups up to lactation day 4 revealed no treatment-related changes. In addition, the ano-genital distance and ano-genital ratio were not altered and male pups did not exhibit areola/nipple retention until postnatal day 13 at any of the doses tested. In conclusion, the treatment did not induce any tests item-related change in parental body weights, organ weights/ratios, thyroid hormone profile, gross pathology and histopathology in adult animals and pups at any tested dose. Therefore, due to the lack of adverse effects in this study, the NOAEL for developmental toxicity for Wistar rats was considered to be ≥ 1000 mg test item/kg bw/day.

 

Finally, ethylene diacetate (CAS 111 -55 -7) was tested for its teratogenic potential in a prenatal developmental toxicity study in rats according to OECD guideline 414 and in compliance with GLP (Eurofins, 2019). Groups of 24 pregnant rats each were orally exposed to test item doses of 100, 300 and 1000 mg/kg bw/day via gavage between gestation days 5 and 19. The test substance was dissolved in corn oil and administered once daily. A similar constituted group received the vehicle and served as control. At day 19 of pregnancy, dams were sacrificed and maternal and fetal animals were examined. Clinical signs of toxicity and treatment-related mortality was not recorded. Body weight gains and food consumption were at levels comparable to those of control animals. Caesarian section was performed for all rats on gestation day 20 and dams were examined for gross pathological changes. No substance-related effects on the number of corpora lutea, implantations and resorptions were observed. Gross evaluation of placentae revealed no findings. In addition, no toxicologically relevant alterations in offspring viability indices were observed. Litter size and weights, the number of fetuses as well as the sex ratio were comparable to vehicle controls. Fetal external, visceral and skeletal observations revealed minor anomalies such as moderate renal pelvis dilation, delayed skeletal ossification and asymmetrical ossification. The effects were not considered treatment-related as they were within the range of historical vehicle control data. Based on these results and under the conditions of this study, a NOAEL for maternal toxicity and fetal developmental toxicity was ≥ 1000 mg test item/kg bw/day in Wistar rats.

Justification for classification or non-classification

The available data on toxicity to reproduction and developmental toxicity do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.