Diethoxymethane

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on the results of a reproduction/developmental toxicity screening study performed with ethylal and following OECD TG 421 and GLP, the NOAEL was 1000 mg/kg/day, corresponding to the highest tested concentration.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 November 2017 - 07 September 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)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Justification for study design:

The purpose of this study was a screening test for reproductive/developmental effects, with administration of the test item, Ethylal, by oral gavage administration to Sprague Dawley Rats for at least four weeks.
The doses used in this study (0, 300, 600 and 1000 mg/kg/day) were selected in conjunction with the Sponsor. Dose levels were selected following the completion of the preliminary toxicity study (Envigo Study number: WG36YM).
In that study, dose levels of 300, 600 and 1000 mg/kg/day were well tolerated with no clinical signs or abnormal macroscopic findings and there were no adverse effects on food intake or body weight gain. There were initial post dosed signs of decreased activity in all animals receiving 1000 mg/kg/day, and unsteady gait in all males and 2/3 females at this dose level. Decreased activity, unsteady gait and piloerection was observed in one male receiving 1000 mg/kg/day on Day 2, after which no post dose signs were observed.
Based on the data generated on WG36YM and as the LD50 had been reported as 3536 mg/kg bw, a high dose level of 1000 mg/kg/day was selected for use on this study. The intermediate and low dose remained at 600 and 300 mg/kg/day to allow for assessment of a dose response.

Specific details on test material used for the study:

Colourless liquid.
Batch number: 1709141400R

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd
- Age at study initiation: Males 71 to 77 days old. Females 85 to 91 days old.
- Weight at study initiation: Males 336 to 418 g. Females 264 to 321 g.
- Housing: On arrival and non-selective allocation to cages.
- Diet: SDS VRF1 Certified pelleted diet. ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes; ad libitum
- Acclimation period: Males: eight days prior to the commencement of treatment. Females: 22 days prior to the commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): and Humidity (%): Monitored and maintained within the range of 20-24ºC and 40-70%.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The required amount of test item was mixed with a small quantity of the vehicle. Approximately 20% of the final volume of vehicle was gradually added. The formulation was quantitatively transferred and diluted to final volume and magnetically stirred in a sealed container of appropriate size ensuring there is no head space.
Frequency of preparation: Weekly.
Storage of formulation Refrigerated: (2-8°C) in amber glass.

Details on mating procedure:

Pairing commenced - After a minimum of two weeks of treatment.
Male/female ratio - 1:1 from within the same treatment groups.
Duration of pairing - Up to two weeks.
Daily checks for evidence of mating - Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation - When positive evidence of mating was detected.
Male/female separation Day - when mating evidence was detected.
Pre-coital interval - Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical procedure was successfully validated for Ethylal in corn oil with respect to the specificity of chromatographic analysis, limit of detection and quantification, the linearity of detector response, precision of injection, method accuracy and precision, standard solution stability and extracted sample solution stability.
The mean concentrations of Ethylal in test formulations analyzed for the study were within +10/-15% of nominal concentrations, confirming accurate formulation. With the exception of Week 4 Group 2, which was confirmed at -20.8%. The difference from mean and coefficient of variation remained within 5%, confirming precise analysis.
(Table 2)

Duration of treatment / exposure:

Males: Two weeks pre-pairing up to necropsy after minimum of four weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 12 of lactation.
Animals of the F1 generation were not dosed.

Frequency of treatment:

Once daily at approximately the same time each day.

Details on study schedule:

- parental animals not mated until 2 weeks after selection

- Age at mating of the mated animals in the study: Males 78 to 84 days old. Females 92 to 98 days old.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control - corn oil

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

600 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on preliminary toxicity study.
- Animal allocation: On arrival and non-selective allocation to cages. Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study. On Day 1 of study all animals were weighed and body weights were reviewed by Study Management before dosing commenced to ensure variations in body weight of animals did not exceed ±20% of the mean for each sex.

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary. A complete necropsy was performed in all cases.

CLINICAL OBSERVATIONS: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
A detailed physical examination was performed on each animal to monitor general health according to the following schedule:
F0 - males Weekly
F0 - females Week 1 and 2 - weekly
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 13

BODY WEIGHT:
The weight of animals was recorded as follows:
F0 males - Weekly during acclimatization (not reported)
Before dosing on the day that treatment commenced (Day 1), and weekly thereafter.
On the day of necropsy.

F0 females - Weekly during acclimatization (not reported)
Before dosing on the day that treatment commenced (Day 1) and weekly before pairing.
Days 0, 7, 14 and 20 after mating.
Day 1, 4, 7 and 13 of lactation.
On the day of necropsy.

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals: Weekly before pairing, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Days 15 to 22), but recommenced for males in Week 4.
For females after mating food consumption was recorded as follows:
Days 0-6, 7-13 and 14-19 after mating
Days 1-3, 4-6 and 7-12 of lactation.
From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.


SIGNS ASSOCIATED WITH DOSING
Detailed observations were recorded at the following times in relation to dose administration and according to the following schedule:
F0 males: Week 1 - daily
Week 2 onwards - twice each week

F0 females: Week 1 - daily
Week 2 - twice
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 12

Detailed observations were recorded at the following times in relation to dose administration:
Pre-dose observation
One to two hours after completion of dosing all groups
As late as possible in the working day.

Oestrous cyclicity (parental animals):

Dry and wet smears were taken as follows:
Dry smears: For 15 days before pairing using cotton swabs.

Wet smears: Using pipette lavage during the following phases:
For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
After pairing until mating.
For four days before scheduled termination.

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight, epididymis weight, sperm morphology

Litter observations:

From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Records Made During Littering Phase:
Clinical observations - Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size - Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter - Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights - Days 1, 4, 7, 11 and 13 of age.
Ano-genital distance - Day 1 - all F1 offspring.
Nipple/areolae count - Day 13 of age - male offspring.

Postmortem examinations (parental animals):

Abnormalities, Epididymides (caput, corpus and cauda), Ovaries, pituitary, prostate, seminal vesicles, testes, thyroids, uterus with cervix and oviducts.
For females the following were recorded:
Each uterine horn, number of implantation sites was counted and confirmed if none were visible at visual inspection.

The organs weighed, tissue samples fixed and sections examined microscopically are detailed below in table 1 for F0 animals:

Postmortem examinations (offspring):

Premature deaths: Where possible, a fresh external macroscopic examination with an assessment of stomach for milk content. Abnormal pups were retained.
F1 offspring on Day 4 of age: Blood sampling required.
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia.
F1 offspring on Day 13 of age: Blood sampling required.
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia.
Thyroid glands were preserved from two offspring per litter, one male and one female in each litter, where possible.

Statistics:

A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level.
If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead.

A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pretreatment data, Kruskal-Wallis’ test was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests were made.
If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead.

For live birth and viability indices dichotomized to 1 when 100% and 0 otherwise, if the Cochran-Armitage test was significant at the 5% level, then the direction of the trend was established and one-tailed step-down testing in this direction was performed. If the Cochran-Armitage test was not significant at the 5% level, then a Chi-square test was applied. If the Chi-square test was significant at the 5% level, the treatment groups were compared using pairwise comparisons of each dose group against the Control using Fisher’s exact tests; otherwise, no further comparisons were made.

For gestation length an exact two-tailed Linear-by-linear test, with equally spaced scores, was applied to all groups.

For estrous cycles an exact one-tailed (upper-tail) Linear-by-linear test was applied to all groups, using scores appropriate to the severity of the observation assuming 4 day cycles to be normal.

For organ weight data, analysis of covariance was performed using terminal body weight as covariate, unless non-parametric methods were applied.

Reproductive indices:

Estrous Cycle, Pre-Coital Interval, Mating Performance and Fertility, Gestation Length and Index,

Offspring viability indices:

Litter Size, Survival Indices, sex ratio

Clinical signs:

no effects observed

Description (incidence and severity):

See tables 1-3

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One group 2 female (Animal No. 134) was found dead on Day 23 of gestation. The animal did not have any adverse clinical signs prior to death, and there were no findings at macroscopic examination, but the female gave birth to 6 pups which were killed for welfare reasons.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The overall bodyweight gain (Day 1-29) and bodyweight change during treatment was statistically significantly lower in males that received 1000 mg/kg/day when compared with Controls, this was predominantly due to statistically significantly reduced weight gain in the first week of treatment.
The overall body weight gain during lactation (Days 1-13) in all females treated with Ethylal, was statistically significantly lower when compared with the Controls this was predominantly due to minor weight loss on Days 1-4 at 300 or 1000 mg/kg/day and statistically significantly reduced weight gain on Days 7-13 of lactation at all doses. However, there was no dose response.
See tables 4-6

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food intake was marginally but statistically significantly reduced during Days 1-8 prior to pairing for females receiving 1000 mg/kg/day when compared with Controls. However, food intake was statistically significantly higher for females receiving Ethylal during Day 14-20 of gestation and lower for females receiving 600 or 1000 mg/kg/day during Days 7-13 of lactation, but a dose response was not apparent.
See tables 7-9

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:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Tables 24-25

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

See tables 10-11

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

For precoital interval, mating performance & fertility, and gestation length & gestation index see tables 12-14.
See table 26 for thyroid hormone (T4) results.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive performance

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

testes

other: epididymides

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

female reproductive system

Organ:

ovary

uterus

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

See table 19

Food consumption and compound intake (if feeding study):

not examined

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:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

See table 26 for thyroid hormone (T4) results.

See tables 15-19

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

haematology

gross pathology

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

endocrine system

Organ:

thyroid gland

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

no

         

 

	Control	Ethylal
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	300	600	1000

 

Table 1: Clinical signs – group distribution of observations for males and for females before pairing (F0)

			Number of animals affected
Category	Observation	Group/Sex : Initial no :	1M 10	2M 10	3M 10	4M 10	1F 10	2F 10	3F 10	4F 10
Behavior	Aggressive		0	0	1	2	0	0	0	0
	Grinding teeth		1	0	0	0	0	0	0	0
	Vocalization		0	0	0	2	0	0	0	0
Body Temperature	Abnormally cold to touch		0	0	0	0	0	1	0	0
Breathing	Irregular		0	0	0	0	0	1	0	0
Coat	Hair loss, Dorsal surface		0	0	0	0	0	0	0	1
	Hair loss, Forelimbs		0	1	0	0	0	0	0	0
	Ungroomed		1	0	0	0	0	0	0	0
Eyes	Pale, Bilateral		0	0	0	0	0	1	0	0
Muscle reaction	Abnormal gait, Uncoordinated		0	0	0	0	0	1	0	0
	Impaired locomotion, Other		0	0	0	0	0	1	0	0

 

Table 1 (cont): Clinical signs – group distribution of observations for males and for females before pairing (F0)

			Number of animals affected
Category	Observation	Group/Sex : Initial no :	1M 10	2M 10	3M 10	4M 10	1F 10	2F 10	3F 10	4F 10
Reason for dispatch	Loss of or limited locomotion		0	0	0	0	0	1	0	0
Skin	Cut, Lower ventral surface		0	0	0	0	0	0	1	0
	Encrustation, Dorsal surface		0	0	0	0	0	2	0	0
	Encrustation, Forelimb - left		0	1	0	0	0	0	0	0
	Encrustation, Lower ventral surface		0	0	0	0	0	0	1	0
	Encrustation, Upper dorsal surface		1	0	0	0	0	0	0	1
Skin color	Reddening, Hindlimbs		1	0	0	0	0	0	0	0
Staining	Abnormal color, Brown, Eye - left		0	0	2	1	0	0	0	0
	Abnormal color, Brown, Head		0	0	0	0	0	0	1	1
	Abnormal color, Brown, Muzzle		1	0	0	1	0	0	0	0
	Abnormal color, Red, Muzzle		1	0	0	0	0	0	0	0
Staining	Chromodacryorrhea		0	0	1	1	0	0	0	0

                                       

Table 2: Clinical signs – group distribution of observations for females during gestation (F0)

			Number of animals affected
Category	Observation	Group/Sex : Initial no :	1F 10	2F 9	3F 10	4F 10
Behavior	Decreased activity		0	0	2	0
Breathing	Irregular		0	0	1	0
	Slow		0	0	1	0
Build (Deformity)	Prominent area, Ventral surface		1	0	0	1
Coat	Hair loss, Dorsal surface		0	0	0	1
	Hair loss, Forelimbs		0	0	0	6
	Hair loss, Ventral surface		0	0	0	1
	Piloerection		0	0	2	0
Eyes	Dark, Bilateral		0	0	1	0
	Dull, Bilateral		0	0	1	0
Reason for dispatch	General poor clinical condition		0	0	2	0

 

Table 2 (cont): Clinical signs – group distribution of observations for females during gestation (F0)

			Number of animals affected
Category	Observation	Group/Sex : Initial no :	1F 10	2F 9	3F 10	4F 10
Skin	Encrustation, Dorsal surface		0	1	0	0
	Encrustation, Ears		0	1	0	0
	Encrustation, Forelimb digit(s)		0	0	1	0
	Encrustation, Upper dorsal surface		0	0	0	2
Skin color	Pallor, Whole body		0	0	2	0
Staining	Abnormal color, Brown, Ears		1	0	1	0
	Abnormal color, Brown, Head		2	4	2	4
	Abnormal color, Brown, Upper dorsal surface		6	3	1	1
	Chromodacryorrhea		2	0	2	0

 

Table 3: Clinical signs – group distribution of observations for females during lactation (F0)

			Number of animals affected
Category	Observation	Group/Sex : Initial no :	1F 10	2F 8	3F 8	4F 10
Behavior	Vocalization		0	0	0	1
Build (Deformity)	Partially absent appendage, Ear - right		0	1	0	0
	Prominent area, Ventral surface		7	6	8	8
Coat	Hair loss, Dorsal surface		0	0	0	1
	Hair loss, Forelimbs		0	2	1	5
	Hair loss, Hindlimbs		0	1	1	3
	Hair loss, Ventral surface		0	1	1	1
Staining	Abnormal color, Brown, Ears		0	0	1	0
	Abnormal color, Brown, Head		1	2	2	2
	Abnormal color, Brown, Upper dorsal surface		8	4	4	3

                                          

Table 4: Body weight and body weight change – group mean values (g) for males and for females before pairing (F0)

Group /Sex		Day 1	8	15	22	29	Change 1-8	Change 8-15	Change 15-22	Change 22-29	Change 1-29
Statistics test		Av	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi
1M	Mean	373	399	422	434	461	26	22	13	26	87
	SD	24.5	32.7	36.6	40.5	43.4	9.8	6.0	6.9	5.0	20.9
	N	10	10	10	10	10	10	10	10	10	10
2M	Mean	378	405	430	446	473	28	25	16	27	96
	SD	17.3	22.1	23.3	26.7	30.4	5.6	4.6	5.6	5.2	15.0
	N	10	10	10	10	10	10	10	10	10	10
3M	Mean	370	394	416	428	453	24	22	12	25	82
	SD	17.0	21.1	26.2	25.2	26.4	8.0	6.6	4.5	5.2	13.8
	N	10	10	10	10	10	10	10	10	10	10
4M	Mean	373	390	409	418	438	16**	19	9	20*	65**
	SD	19.2	19.2	21.0	21.0	20.7	6.3	8.4	4.8	6.5	12.0
	N	10	10	10	10	10	10	10	10	10	10

 

Table 4 (cont): Body weight and body weight change – group mean values (g) for males and for females before pairing (F0)

Group /Sex		Day 1	8	15	Change 1-8	Change 8-15	Change 1-15
Statistics test		Av	Wi	Wi	Wi	Wi	Wi
1F	Mean	290	293	294	3	1	5
	SD	16.7	16.5	18.5	3.6	8.1	9.3
	N	10	10	10	10	10	10
2F	Mean	291	292	301	1	9	11
	SD	10.0	9.9	9.4	7.5	6.8	8.4
	N	10	10	9	10	9	9
3F	Mean	294	296	305	2	9	11
	SD	16.3	16.7	17.8	4.2	6.1	5.8
	N	10	10	10	10	10	10
4F	Mean	296	296	301	1	4	5
	SD	12.3	11.4	10.4	4.0	7.3	8.5
	N	10	10	10	10	10	10

 

Table 5: Body weight and body weight change – group mean values (g) for females during gestation (F0)

Group /Sex		Day 0	7	14	20	Change 0-7	Change 7-14	Change 14-20	Change 0-20
Statistics test		Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi
1F	Mean	304	328	360	441	24	33	80	137
	SD	16.3	15.0	12.0	12.8	8.0	6.9	12.4	15.5
	N	10	10	10	10	10	10	10	10
2F	Mean	305	334	369	456	29	36	87	151
	SD	13.6	7.6	11.1	14.0	9.1	8.5	9.4	14.3
	N	9	9	9	9	9	9	9	9
3F	Mean	308	334	366	448	26	32	82	140
	SD	17.5	21.0	20.5	26.7	9.4	7.8	11.5	18.8
	N	10	10	10	10	10	10	10	10
4F	Mean	300	328	367	448	28	39	81	148
	SD	13.5	8.7	13.6	20.9	9.7	8.3	10.8	16.5
	N	10	10	10	10	10	10	10	10

Table 6: Body weight and body weight change – group mean values (g) for females during lactation (F0)

Group /Sex		Day 1	4	7	13	Change 1-4	Change 4-7	Change 7-13	Change 1-13
Statistics test		Wi	Wi	Wi	Wi	Sh	Wi	Wi	Wi
1F	Mean	339	341	358	382	2	17	25	44
	SD	14.3	16.6	15.8	16.0	6.9	9.3	8.2	13.3
	N	10	10	10	10	10	10	10	10
2F	Mean	346	344	360	369	-2	15	9*	23*
	SD	12.4	5.7	8.4	8.0	11.3	4.3	8.9	14.8
	N	8	8	8	8	8	8	8	8
3F	Mean	345	351	360	374	6	9	14*	29*
	SD	22.1	22.8	21.9	15.8	8.9	7.5	9.8	12.1
	N	8	8	8	8	8	8	8	8
4F	Mean	347	345	358	376	-2	13	18**	29*
	SD	18.8	18.3	15.8	13.7	24.4	8.9	6.3	18.4
	N	10	10	10	10	10	10	10	10

                               

Table 7: Food consumption – group mean values (g/animal/day) for males and for females before pairing (F0)

Group /Sex		Day 1-8	8-15	15-22	22-29
Statistics test		Wi	Wi		Wi
1M	Mean	22	20	#	20
	SD	1.3	1.3		1.0
	N	3	3		3
2M	Mean	23	21	#	22
	SD	1.2	0.9		1.1
	N	3	3		3
3M	Mean	22	20	#	21
	SD	1.2	1.1		1.2
	N	3	3		3
4M	Mean	20	20	#	21
	SD	1.2	0.9		1.0
	N	3	3		3

# - Food consumption not recorded whilst paired for mating

Table 7 (cont): Food consumption – group mean values (g/animal/day) for males and for females before pairing (F0)

Group /Sex		Day 1-8	8-15
Statistics test		Wi	Wi
1F	Mean	15	14
	SD	0.7	1.0
	N	3	3
2F	Mean	14	15
	SD	0.7	0.7
	N	3	3
3F	Mean	15	15
	SD	0.6	0.1
	N	3	3
4F	Mean	14*	14
	SD	0.4	0.6
	N	3	3

Table 8: Food consumption – group mean values (g/animal/day) for females during gestation (F0)

Group /Sex		Day 1-7	7-14	14-20
Statistics test		Wi	Wi	Wi
1F	Mean	19	21	22
	SD	1.7	3.7	3.9
	N	10	10	10
2F	Mean	19	20	25*
	SD	1.1	4.8	2.0
	N	9	8	9
3F	Mean	19	21	24*
	SD	1.9	2.1	1.5
	N	10	10	10
4F	Mean	19	22	26**
	SD	1.7	1.7	2.5
	N	10	10	10

                                      

Table 9: Food consumption – group mean values (g/animal/day) for females during lactation (F0)

Group /Sex		Day 1-4	4-7	7-13
Statistics test		Wi	Wi	Wi
1F	Mean	30	51	70
	SD	5.0	4.3	4.1
	N	10	10	10
2F	Mean	35	50	66
	SD	3.7	5.0	3.1
	N	8	8	8
3F	Mean	32	48	64*
	SD	6.1	5.7	8.5
	N	8	8	8
4F	Mean	30	46	63*
	SD	9.4	7.1	5.4
	N	10	10	10

Table 10: Estrous cycles – group values before treatment (F0)

Group	Number of Animals		4 day	Regular cycles   4/5 days	5 day	Irregular cycle*	Extended estrus**	Acyclicy***
1	10	N (%)	9 (90)	1 (10)	0	0	0	0
2	10	N (%)	8 (80)	1 (10)	1 (10)	0	0	0
3	10	N (%)	7 (70)	1 (10)	2 (20)	0	0	0
4	10	N (%)	9 (90)	1 (10)	0	0	0	0

* At least one cycle of two, three or six to ten days

** At least four consecutive days of estrus

*** At least ten days without estrus

Table 11: Estrous cycles – group values during treatment (F0)

Group	Number of Animals		4 day	Regular cycles   4/5 days	5 day	Irregular cycle*	Extended estrus**	Acyclic***
Statistics test	Lt
1	10	N (%)	9 (90)	1 (10)	0	0	0	0
2	10	N (%)	8 (80)	1 (10)	0	1 (10)	0	0
3	10	N (%)	8 (80)	0	1 (10)	1 (10)	0	0
4	10	N (%)	6 (60)	2 (20)	0	1 (10)	1 (10)	0

 

 

Table 12: Pre-coital interval – group values (F0)

			Pre-coital interval (days)
Group	Number of animals		1-4	5-8	9-12	13-14
1	10	N (%)	10 (100)	0	0	0
2	9	N (%)	9 (100)	0	0	0
3	10	N (%)	10 (100)	0	0	0
4	10	N (%)	10 (100)	0	0	0

                             

Table 13: Mating performance and fertility – group values (F0)

Group and sex	Number paired	Number mating	Number achieving pregnancy	Percentage mating	Conception rate (%)	Fertility index (%)
1M	10	10	10	100	100	100
2M	9	9	9	100	100	100
3M	10	10	10	100	100	100
4M	10	10	10	100	100	100
1F	10	10	10	100	100	100
2F	9	9	9	100	100	100
3F	10	10	10	100	100	100
4F	10	10	10	100	100	100

 

Table 14: Gestation length and gestation index – group values (F0)

			Gestation length (days)
Group	Number of pregnant animals		22	22.5	23	23.5	Number of liver litters born	Gestation index (%)
Statistics test			Lt					CA
1	10	N (%)	1 (10)	6 (60)	3 (30)	0	10	100
2	9	N (%)	0	2 (22)	7 (78)	0	8A	89
3	10B	N (%)	2 (22)	4 (44)	2 (22)	1 (11)	8A	80
4	10	N (%)	0	5 (50)	4 (40)	1 (10)	10	100

A            Excludes one female which did not produce a complete live litter

B            Percentage distribution of gestation lengths calculated from nine animals - one pregnant female failed to litter, killed for welfare reasons on Day 24 of gestation

Table 15: Litter size – group mean values (F1)

Group /Sex		Implantations	Total @	Live on Day Before bleed 1	4	After bleed 4	13
Statistics test		Wi	Wi	Wi	Wi
1F	Mean	15.6	14.4	14.0	13.9	12.5	12.5
	SD	2.55	2.41	2.21	2.18	1.78	1.78
	N	10	10	10	10	10	10
2F	Mean	17.0	15.8	15.0	15.0	13.3	13.3
	SD	1.41	1.28	1.60	1.60	1.91	1.91
	N	9	8	8	8	8	8
3F	Mean	15.5	14.9	13.5	13.3	11.9	11.9
	SD	3.03	3.48	3.21	3.15	2.42	2.42
	N	10	8	8	8	8	8
4F	Mean	16.2	14.8	13.4	13.1	11.6	11.6
	SD	2.35	2.74	2.50	2.64	2.07	2.07
	N	10	10	10	10	10	10

@ - Includes offspring that died prior to the designated Day 1 of age

Table 16: Offspring survival indices – group mean values (F1)

Group /Sex		Post implantation survival index (%)	Live birth index (%)	Viability index (%) Day 4	Lactation index (%) Day 13
Statistics test		Wi	Ch	Ch
1F	Mean	92.2	97.5	99.3	100.0
	SD	8.04	4.30	2.11	0.00
	N	10	10	10	10
	N<100%	6	3	1	0
2F	Mean	94.1	95.1	100.0	100.0
	SD	4.55	4.42	0.00	0.0
	N	8	8	8	8
	N<100%	6	5	0	0
3F	Mean	93.8	91.1	98.3	100.0
	SD	8.89	11.31	3.20	0.00
	N	8	8	8	8
	N<100%	3	4	2	0
4F	Mean	91.1	91.2	97.5	100.0
	SD	9.41	10.25	4.17	0.00
	N	10	10	10	10
	N<100%	7	6	3	0

The following data were used for the statistics tests, animal indices for post implantation survival index and animal indices dichotomized to 1 when 100% and 0 otherwise for live birth and viability indices.

 

Table 17: Offspring sex ratio – group mean values

Group /Sex		Total @			Live (before bleed) on Day						Live (after bleed) on Day
		M	F	%M	1 M	F	%M	4 M	F	%M	4 M	F	%M	13 M	F	%M
Statistic test				Wi			Wi			Wi
1F	Mean	8.5	5.7	59.7	8.3	5.7	59.1	8.2	5.7	58.8	8.2	4.3	64.9	8.2	4.3	64.9
	SD	2.32	1.95	12.33	2.26	1.95	12.27	2.30	1.95	12.49	2.30	1.42	12.17	2.30	1.42	12.16
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
2F	Mean	7.6	8.0	48.3	7.3	7.8	47.6	7.3	7.8	47.6	7.3	6.0	53.5	7.3	6.0	53.5
	SD	2.72	2.20	15.05	2.87	2.12	15.43	2.87	2.12	15.43	2.87	1.51	13.78	2.87	1.51	13.78
	N	8	8	8	8	8	8	8	8	8	8	8	8	8	8	8
3F	Mean	6.8	8.0	46.4	6.5	7.0	47.9	6.4	6.9	47.7	6.4	5.5	52.8	6.4	5.5	52.8
	SD	1.91	3.07	12.89	2.07	2.07	10.31	2.07	1.96	10.06	2.07	1.31	11.00	2.07	1.31	11.00
	N	8	8	8	8	8	8	8	8	8	8	8	8	8	8	8
4F	Mean	7.2	6.9	51.7	7.0	6.4	52.7	6.8	6.3	52.5	6.8	4.8	58.8	6.8	4.8	58.8
	SD	1.99	2.56	13.62	2.05	2.41	13.42	1.93	2.41	12.65	1.93	1.99	13.65	1.93	1.99	13.65
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10

@- Includes offspring that died prior to the designated Day 1 of age and excludes unsexed offspring

Table 18: Ano-genital distance – group mean absolute and adjusted values for offspring (F1)

Group /Sex		Body weight (g) Day 1	Ano-genital distance (mm)
Statistics test		Wi
1M	Mean	7.3	4.2
	SD	0.75	0.27
	N	10	10
2M	Mean	7.6	4.5
	SD	0.38	0.16
	N	8	8
3M	Mean	6.9	4.3
	SD	0.65	0.30
	N	8	8
4M	Mean	7.0	4.3
	SD	0.38	0.34
	N	10	10

 

Adjusted means
Statistics test		Wi
1M	Mean	4.2
2M	Mean	4.4
3M	Mean	4.3
4M	Mean	4.4

 

Table 18 (cont): Ano-genital distance – group mean absolute and adjusted values for offspring (F1)

Group /Sex		Body weight (g) Day 1	Ano-genital distance (mm)
Statistics test		Wi
1F	Mean	6.9	2.2
	SD	0.73	0.16
	N	10	10
2F	Mean	7.2	2.2
	SD	0.51	0.08
	N	8	8
3F	Mean	6.5	2.1
	SD	0.72	0.15
	N	8	8
4F	Mean	6.6	2.1
	SD	0.54	0.13
	N	10	10

 

Adjusted means
Statistics test		Wi
1F	Mean	2.2
2F	Mean	2.2
3F	Mean	2.1
4F	Mean	2.1

Table 19: Bodyweight – group mean values (g) for offspring (F1)

Group /Sex		Day of age (before bleed)			Day of age (after bleed)				Change 1-4	Change 4-7	Change 7-11	Change 11-13	Change 1-13
		1	1@	4	4	7	11	13
Statistics test		Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi
1M	Mean	7.3	7.3	10.2	10.2	15.4	24.3	28.8	3.0	5.2	8.9	4.5	21.5
	SD	0.75	0.74	1.34	1.34	2.18	2.94	3.32	0.72	1.01	0.90	0.49	2.75
	N	10	10	10	10	10	10	10	10	10	10	10	10
2M	Mean	7.6	7.6	10.7	10.7	15.8	24.0	27.9	3.1	5.0	8.2	3.9	20.3
	SD	0.38	0.38	1.11	1.11	1.66	3.14	3.86	0.78	0.71	1.51	0.82	3.55
	N	8	8	8	8	8	8	8	8	8	8	8	8
3M	Mean	6.9	6.9	10.2	10.2	15.6	24.1	28.4	3.3	5.4	8.6	4.3	21.6
	SD	0.65	0.65	1.18	1.18	1.87	2.77	3.39	0.83	0.81	1.03	0.76	3.13
	N	8	8	8	8	8	8	8	8	8	8	8	8
4M	Mean	7.0	7.0	10.0	10.0	15.1	23.6	27.9	2.9	5.2	8.5	4.3	20.9
	SD	0.38	0.46	1.13	1.13	1.68	2.25	2.74	0.8	0.85	0.95	0.78	2.47
	N	10	10	10	10	10	10	10	10	10	10	10	10

@ - Includes only those pups surviving after the bleed

Table 19 (cont): Bodyweight – group mean values (g) for offspring (F1)

Group /Sex		Day of age (before bleed)			Day of age (after bleed)				Change 1-4	Change 4-7	Change 7-11	Change 11-13	Change 1-13
		1	1@	4	4	7	11	13
Statistics test		Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi
1F	Mean	6.9	6.9	9.7	9.7	14.7	23.6	27.8	2.8	5.0	8.9	4.3	21.0
	SD	0.73	0.75	1.31	1.31	2.17	3.10	3.52	0.70	0.97	1.00	0.49	2.93
	N	10	10	10	10	10	10	10	10	10	10	10	10
2F	Mean	7.2	7.2	10.2	10.1	15.0	23.0	26.8	2.9	4.9	8.0	3.8	19.6
	SD	0.51	0.50	1.10	1.15	1.86	3.11	4.02	0.77	0.96	1.31	0.95	3.56
	N	8	8	8	8	8	8	8	8	8	8	8	8
3F	Mean	6.5	6.5	9.7	9.7	14.9	23.4	27.6	3.2	5.2	8.5	4.2	21.1
	SD	0.72	0.74	1.36	1.40	2.03	3.39	3.91	0.99	0.80	1.52	0.63	3.52
	N	8	8	8	8	8	8	8	8	8	8	8	8
4F	Mean	6.6	6.6	9.5	9.5	14.5	22.6	26.7	2.9	4.9	8.1	4.0	20.0
	SD	0.54	0.52	1.32	1.31	1.83	2.68	3.04	0.87	0.70	1.10	0.79	2.72
	N	10	10	10	10	10	10	10	10	10	10	10	10

@ - Includes only those pups surviving after the bleed

Table 20: Organ weights – group mean absolute and adjusted values (g) for males killed at scheduled termination (F0)

Group /Sex		Terminal Body weight	Epididymides	Prostate	Seminal vesicles with coagulating gland	Testes
Statistics test		Wi
1M	Mean	459	1.245	1.120	2.129	3.542
	SD	43	0.110	0.231	0.253	0.232
	N	10	10	10	10	10
2M	Mean	471	1.253	1.047	2.208	3.550
	SD	30	0.078	0.180	0.247	0.197
	N	10	10	10	10	10
3M	Mean	450	1.249	1.040	2.109	3.708
	SD	27	0.109	0.213	0.255	0.379
	N	10	10	10	10	10
3M	Mean	436	1.222	0.961	2.127	3.482
	SD	21	0.091	0.147	0.382	0.300
	N	10	10	10	10	10

 

Statistics test		Wi	Wi	Wi	Wi
1M	Adjusted mean	1.237	1.107	2.111	3.523
2M	Adjusted mean	1.225	1.003	2.146	3.488
3M	Adjusted mean	1.255	1.050	2.123	3.722
4M	Adjusted mean	1.252	1.008	2.193	3.549

                  

Table 21: Organ weights – group mean absolute and adjusted values (g) for females killed on Day 13 of lactation (F0)

Group /Sex		Terminal Body weight	Ovaries	Uterus and Cervix and Oviducts
Statistics test		Wi
1F	Mean	382	0.097	0.503
	SD	16	0.007	0.129
	N	10	10	10
2F	Mean	371	0.088	0.423
	SD	8	0.010	0.044
	N	8	8	8
3F	Mean	375	0.090	0.434
	SD	17	0.008	0.050
	N	8	8	8
4F	Mean	376	0.086	0.480
	SD	15	0.018	0.061
	N	10	10	10

 

Statistics test		Wi	Wi
1F	Adjusted mean	0.096	0.484
2F	Adjusted mean	0.088	0.426
3F	Adjusted mean	0.090	0.432
4F	Adjusted mean	0.086	0.477

 

Table 22: Macropathology – group distribution of findings for males killed at scheduled termination (F0)

		Number of animals affected
Tissue/Organ and Findings	Group/Sex: No of animals:	1M 10	2M 10	3M 10	4M 10
Number of animals within normal limits		10	10	10	10

 

Table 23: Macropathology – group distribution of findings for females killed on Day 13 of lactation (F0)

		Number of animals affected
Tissue/Organ and Findings	Group/Sex: No of animals:	1F 10	2F 8	3F 8	4F 10
Number of animals within normal limits		9	8	8	9
Skin and Subcutis Hair Loss		0	0	0	1
Uterus Abnormal contents Mass(es)		1 1	0 0	0 0	0 0

 

Table 24: Histopathology – group distribution of findings for males killed at scheduled termination (F0)

		Number of animals affected
Tissue/Organ and Findings	Group/Sex: No of animals:	1M 10	2M 10	3M 10	4M 10
Epididymides Infiltrate, Inflammatory Cell	No. examined Minimal Total	10 2 2	0 0 0	0 0 0	10 3 3
Testes	No. examined	10	0	0	10

 

Table 25: Histopathology – group distribution of findings for females killed on Day 13 of lactation (F0)

		Number of animals affected
Tissue/Organ and Findings	Group/Sex: No of animals:	1F 10	2F 8	3F 8	4F 10
Ovaries	No. examined	10	0	0	10
Uterus Implantation site(s)   Dilatation, Luminal	No. examined Total Minimal Total	1 1 1 1	0 0 0 0	0 0 0 0	0 0 0 0

 

 

Table 26: Mean serum T4 concentrations (pg/mL)

Group	Treatment	Dose (mg/kg/day)		Adult terminal males	Male offspring on Day 13 of age	Female offspring on Day 13 of age
1	Control (Corn Oil)	0	Mean	43300	50700	59200
			SD	7610	10800	14400
			CV %	17.6	21.3	24.3
			N	10	10	10
2	Ethylal	300	Mean	46000	56500	51500
			SD	7590	18300	6230
			CV %	16.5	32.4	12.1
			N	10	8	8
3	Ethylal	600	Mean	47100	55700	63200
			SD	7000	15800	14800
			CV %	14.9	28.4	23.4
			N	10	8	8
4	Ethylal	1000	Mean	44300	59300	46400
			SD	5960	10600	12400
			CV %	13.5	17.9	26.7
			N	10	10	10

Conclusions:

It was concluded that in the absence of any evidence for general systemic toxicity or effects on reproductive performance/offspring development that the no observed adverse effect level (NOAEL) for Ethylal was 1000 mg/kg/day.

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:

Endpoint derived from an OECD TG 421 (GLP) study with ethylal. RSS of the test report is provided.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Based on the results of a prenatal developmental toxicity study performed with ethylal and following OECD TG 414 and GLP, the maternal and the embryo-fetal NOAEL was 1000 mg/kg/day, corresponding to the highest tested concentration.

Based on the results of a reproduction/developmental toxicity screening study performed with ethylal and following OECD TG 421 and GLP, the NOAEL was 1000 mg/kg/day, corresponding to the highest tested concentration.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14 NOVEMBER 2018 to 22 FEBRUARY 2019

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:

03 September 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Lambiotte & Cie - 1808061400R
- Expiration date of the lot/batch: 06 August 2020

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd
- Age at study initiation: Approximately 71 days old
- Weight at study initiation: 228 to 290 g
- Housing: Acclimatization: up to four animals per cage; During pairing: one (stock) male and one female per cage; Gestation: one female per cage
- Diet: SDS VRF1 Certified pelleted diet. Non-restricted
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Non-restricted
- Acclimation period: Five days before commencement of pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod: 12 hours light: 12 hours dark

IN-LIFE DATES: From: 14 November 2018 To:10 to 15 December 2018

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Starting with the lowest concentration, the required amount of test item was weighed into a suitable container and immediately transferred to a measuring cylinder. Vehicle was added to make up to about 20% of the final volume. The weighing container was rinsed out and formulation made up to the required volume with vehicle.

The formulation was then magnetically stirred in a sealed container of the appropriate size with no head space.

A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.

Frequency of preparation: Weekly.

Storage of formulation: Refrigerated (2 to 8 ºC) for up to 10 days.

VEHICLE
- Concentration in vehicle: 0, 20, 66 and 200 mg/mL
- Amount of vehicle: 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method involved extraction and dilution in acetone followed by gas chromatographic analysis with flame ionization detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 10 μg/mL to 100 μg/mL.

The mean concentrations were within +10/-15% of the nominal concentration, confirming accurate formulation. The percentage difference from mean values remained within 1%, confirming precise analysis, with the exception of the last formulation occasion of Group 4. Initial results from the last sample occasion of Group 4 were low; however, extracted samples were reanalyzed, in accordance with standard operating procedures, confirming the mean concentrations were within acceptance criteria. As a result, the percentage difference for this group was expectedly high (±10.49%).

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1 with identified stock males
- Daily checks for evidence of mating: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
- Day 0 of gestation: When positive evidence of mating was detected.
A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.

Duration of treatment / exposure:

Females: Day 6 to 19 after mating

Frequency of treatment:

Once daily at approximately the same time each day

Duration of test:

20 days

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

330 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control - corn oil

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Dose levels have been selected, in conjunction with the Sponsor, based on the effects seen in a preliminary embryo-fetal study conducted at this laboratory which investigated dose levels of 330, 660 and 1000 mg/kg/day. Initial treatment from Gestation day 6 to 7 resulted in overall group mean body weight stasis at 330 or 1000 mg/kg/day; however overall body weight gains over the whole treatment period were unaffected. There was no conclusive effect of treatment on embryo-fetal development, survival, litter size or sex ratio at any dose level.
Dose levels of 100, 330 and 1000 mg/kg/day were therefore considered appropriate for this study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

CLINICAL OBSERVATIONS: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.


BODY WEIGHT: The weight of each adult was recorded on Days 0, 3, 6-20 after mating.

FOOD CONSUMPTION: The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive after mating.

SIGNS ASSOCIATED WITH DOSING: Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration:
Pre-dose observation.
Immediately after completion of dosing each Group.
One to two hours after completion of dosing.
As late as possible in the working day.


POST-MORTEM EXAMINATIONS: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Ovaries and uterine content:

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

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes
- Fetal weight: Yes
- Ano-genital distance: Yes

Statistics:

A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pre treatment data, Kruskal-Wallis’ test was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests were made.
If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead.

For live birth and viability indices dichotomized to 1 when 100% and 0 otherwise, if the Cochran Armitage test was significant at the 5% level, then the direction of the trend was established and one-tailed step-down testing in this direction was performed. If the Cochran-Armitage test was not significant at the 5% level, then a Chi-square test was applied. If the Chi-square test was significant at the 5% level, the treatment groups were compared using pairwise comparisons of each dose group against the Control using Fisher’s exact tests; otherwise, no further comparisons were made.
For organ weight data, analysis of covariance was performed using terminal body weight as covariate, unless non-parametric methods were applied.

Indices:

Reproductive assessment:
The following were recorded for all animals:
For each ovary/uterine horn - Number of:
Corpora lutea.
Implantation sites.
Resorption sites (classified as early or late).
Fetuses (live and dead).

Historical control data:

Historical control data on :
- Fetal examinations - Major abnormalities (Table 16)
- Fetal examinations - Skeletal (Table 18)
- Fetal examinations - Visceral (Table 20)
- Thyroid hormone data (Table 22)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At routine physical examination, there was an increased incidence of hair loss of the forelimbs in females treated with Ethylal, although it is unclear if this is treatment related since this finding occurs commonly.
One female (2F 28) was seen with two swollen areas of the upper ventral surface on Day 16, one of which apparently increased in size to 20 x 20 mm.
Following initial treatment (gestation day 6), signs associated with dosing Ethylal at 1000 mg/kg/day included unsteady gait in 11 animals, decreased activity in 5 animals, flattened posture, or gait, in 4 animals and partially closed eye lids in 3 animals.
The type of observation seen was associated with specific signs (i.e. unsteady gait at the start of treatment and chin rubbing towards the end) and although treatment related, the signs were either recoverable or transient and, therefore, considered non-adverse. Signs associated with dosing in Group 3 were confined to salivation which is not viewed as adverse.
Tables 1 and 2

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no clear difference in the overall (gestation day 6 to 20) body weight change for females treated with Ethylal at any dose level.
Body weight loss/stasis was seen in females receiving 330 or 1000 mg/kg/day following initial treatment gestation day 6 to 7, however the body weight performance improved and daily gains were generally similar to Control thereafter.
It is noted that body weight gains from gestation day 0 to 6 (prior to treatment) were statistically significantly low in females allocated to the 1000 mg/kg/day group and consequently not due to treatment.
Body weight gains (gestation days 6 to 20), adjusted for gravid uterine weights were slightly low at 1000 mg/kg/day when compared to Control and, also, achieved statistical significance. However
the overall performance was unaffected by treatment.
Tables 3 and 4

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of females treated at 1000 mg/kg/day was statistically significantly low prior to the start of treatment and remained low throughout treatment, although statistical significance was not attained on the last recording occasion.
Table 5

Food efficiency:

not specified

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

Description (incidence and severity):

Thyroid and parathyroid weights were unaffected by treatment.
There was no effect of treatment on gravid uterine weight
Table 6

Gross pathological findings:

no effects observed

Description (incidence and severity):

Table 7

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The microscopic examination of the thyroids revealed no test item-related lesions.
The incidence and distribution of all findings were considered to be unrelated to treatment. Such findings included the follicular cysts in the thyroid tissue, since there was a low incidence and the distribution of cyst findings was unrelated to treatment.
Table 8

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid hormone analysis:

The group mean values of T3 and T4 concentrations in serum for females receiving 330 or 1000 mg/kg/day were slightly low, when compared to Control, with the difference attaining statistical significance.

The group mean values of serum TSH concentrations in females receiving 1000 mg/kg/day (Group 4) was slightly higher, when compared to Control, with the difference attaining statistical significance.

Details on results:

These minor variations in the circulating levels of T3, T4 and TSH (that were within the normal historical control range) were not associated with changes in ano-genital distances and no macroscopic or microscopic changes to the thyroid gland were observed. In the absence of any histopathological correlate and with no evidence of disturbance to the growth or development of fetuses, the small differences in the circulating levels of thyroid hormones in the females receiving 330 or 1000 mg/kg/day, although statistically significant, were considered nonadverse
Tables 9, 10, 11 and 22

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Tables 12 and 13

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Tables 12 and 13

Early or late resorptions:

no effects observed

Description (incidence and severity):

Tables 12 and 13

Dead fetuses:

no effects observed

Description (incidence and severity):

Tables 12 and 13

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

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: absence of effects

Remarks on result:

other: absence of effects

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Table 14

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Tables 12 and 13

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Table 13

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Table 14

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Description (incidence and severity):

Tables 17 and 18

Visceral malformations:

no effects observed

Description (incidence and severity):

Tables 19 and 20

Other effects:

no effects observed

Description (incidence and severity):

There was no effect of treatment on ano-genital distances.
Table 21

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of effects

Remarks on result:

other: absence of effects

Abnormalities:

no effects observed

Developmental effects observed:

no

	Control	Ethylal
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	100	330	1000

 

Table 1: Signs associated with dosing – group distribution of observations after mating

			Number of animals affected
Category	Observation	Group/Sex: Initial no.:	1F 20	2F 20	3F 20	4F 20
Abnormal gait	Flattened		0	0	0	1
	Unsteady		0	0	0	13
Behavior	Chin rubbing		0	0	0	3
	Decreased activity		0	0	0	8
	Salivation		0	0	4	19
Eyelids	Partially closed, Bilateral		0	0	0	3
Posture	Flattened		0	0	0	5

 

Table 2: Clinical signs - group distribution of observations after mating

			Number of animals affected
Category	Observation	Group/Sex: Initial no.:	1F 20	2F 20	3F 20	4F 20
Build (Deformity)	Swollen area, Upper ventral surface		0	1	0	0
Coat	Hair loss, Forelimbs		0	3	5	3
Skin	Encrustation, Ear - left		1	0	0	0
	Encrustation, Forelimb - left		0	0	1	0
Staining	Abnormal color, Red, Forelimb - left		0	0	0	1

 

 

 

Table 3: Body weight and body weight change - group mean values (g) during gestation

Group /Sex		Day 0	3	6	7	8	9	10	11	12	13	14	15	16
Statistics test		Av	Av	Av	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi
1F	Mean	251	271	283	285	291	295	301	307	314	320	327	335	348
	SD	12.2	13.4	12.3	14.3	13.4	13.7	12.9	13.4	14.1	13.7	13.6	15.5	15.6
	N	20	20	20	20	20	20	20	20	20	20	20	20	20
2F	Mean	252	268	279	282	285	290	296	303	310	316	322	330	342
	SD	13.5	13.6	14.2	15.6	14.9	15.3	16.0	17.5	15.5	17.1	18.5	19.5	19.6
	N	20	20	20	20	20	20	20	20	20	20	20	20	20
3F	Mean	250	268	280	280	285	290	297	303	308	316	323	332	344
	SD	10.0	10.9	12.4	13.5	12.7	12.2	12.7	14.7	13.5	15.1	16.1	15.5	16.4
	N	20	20	20	20	20	20	20	20	20	20	20	20	20
4F	Mean	253	267	278	277	281	285*	291	297	304*	309*	315*	324	336
	SD	15.2	14.3	17.4	18.1	18.3	18.2	19.5	19.4	17.6	19.1	19.3	19.9	22.4
	N	19	19	19	19	19	19	19	19	19	19	19	19	19

 

Table 3 (cont): Body weight and body weight change - group mean values (g) during gestation

Group /Sex		Day 17	18	19	20	Change 0-6	Change 6-20
Statistics test		Wi	Wi	Wi	Wi	Av	Wi
1F	Mean	362	379	395	413	32	130
	SD	15.5	17.8	18.4	20.6	6.9	10.9
	N	20	20	20	20	20	20
2F	Mean	356	373	388	405	27*	125
	SD	21.0	22.1	22.6	26.2	7.3	16.2
	N	20	20	20	20	20	20
3F	Mean	359	375	389	404	30	124
	SD	18.7	21.7	24.0	26.5	6.0	18.3
	N	20	20	20	20	20	20
4F	Mean	351	366	381	398	26**	120
	SD	23.6	24.3	27.9	29.8	5.3	17.9
	N	19	19	19	19	19	19

 

Table 4: Gravid uterine weight, adjusted body weight and adjusted body weight change - group mean values (g) on Day 20 of gestation

Group /Sex		Body weight Day 6	Terminal body weight Day 20	Body weight change 6-20	Gravid uterine weight	Adjusted body weight Day 20	Adjusted body weight change 6-20
Statistics test		Av	Wi	Wi	Sh	Wi	Wi
1F	Mean	283	412	130	92.9	319	37
	SD	12.3	20.2	10.8	7.31	17.7	8.9
	N	20	20	20	20	20	20
2F	Mean	279	405	125	91.2	313	34
	SD	14.2	26.0	16.3	11.99	21.2	10.0
	N	20	20	20	20	20	20
3F	Mean	280	405	125	87.7	317	37
	SD	12.4	26.3	18.2	16.68	15.4	9.3
	N	20	20	20	20	20	20
4F	Mean	278	398	120	89.5	308	30*
	SD	17.4	29.7	18.0	13.41	20.0	8.7
	N	19	19	19	19	19	19

 

Table 5: Food consumption - group mean values (g/animal/day) during gestation

Group /Sex		Day 0-3	3-6	6-10	10-14	14-18	18-20
Statistics test		Av	Av	Wi	Wi	Wi	Wi
1F	Mean	21	23	20	22	25	24
	SD	1.7	1.7	1.7	2.0	2.2	2.3
	N	20	20	20	20	20	20
2F	Mean	20*	22	19	21	23	23
	SD	1.9	1.8	1.7	1.9	2.2	2.2
	N	20	20	20	20	20	20
3F	Mean	21	23	20	22	25	23
	SD	1.9	1.9	1.7	1.4	2.0	2.2
	N	20	20	20	20	20	20
4F	Mean	19**	22*	18**	20**	23**	22
	SD	1.4	1.9	1.7	1.7	2.5	3.7
	N	19	19	19	19	19	19

 

Table 6: Organ weights - group mean absolute and adjusted values (g) on Day 20 of gestation

Group /Sex		Terminal Body weight	Thyroids and Parathyroids
Statistics test		Wi
1F	Mean	412	0.012
	SD	20	0.002
	N	20	20
2F	Mean	405	0.012
	SD	26	0.002
	N	20	20
3F	Mean	405	0.013
	SD	26	0.003
	N	20	20
4F	Mean	398	0.012
	SD	30	0.003
	N	19	19

 

		Thyroids and Parathyroids
Statistics test		Wi
1F	Adjusted Mean	0.012
2F	Adjusted Mean	0.011
3F	Adjusted Mean	0.013
4F	Adjusted Mean	0.012

 

Table 7: Macropathology – group distribution of findings on Day 20 of gestation

		Number of animals affected
Tissue/Organ and Findings	Group/Sex No. of animals	1F 20	2F 20	3F 20	4F 19
Liver Mass(es) Pale area(s)		1 0	0 1	0 0	0 0
Palpable Mass Found at necropsy		0	1	0	0
Placenta Enlarged		1	0	0	0

 

Table 8: Histopathology – group distribution of findings on Day 20 of gestation

		Number of animals affected
Tissue/Organ and Findings	Group/Sex No. of animals	1F 20	2F 20	3F 20	4F 19
Thyroids Cyst(s), Ultimobranchial, Prominent     Cyst(s), Follicular	No. examined Total   Total	20 4   0	20 5   0	19 3   0	19 5   2

 

Table 9: Group mean values for T3

Group	Treatment	mg/kg/day		Value
Statistical test				Wi
1	Control	0	Mean (pg/mL)	448
			SD	111
			N	20
2	Ethylal	100	Mean (pg/mL)	456
			SD	97
			N	19
3	Ethylal	330	Mean (pg/mL)	378*
			SD	96
			N	19
4	Ethylal	1000	Mean (pg/mL)	363*
			SD	107
			N	19

Excluding non-pregnant animals.

Table 10: Group mean values for T4

Group	Treatment	mg/kg/day		Value
Statistical test				Wi
1	Control	0	Mean (pg/mL)	15545
			SD	3122
			N	20
2	Ethylal	100	Mean (pg/mL)	17058
			SD	3658
			N	19
3	Ethylal	330	Mean (pg/mL)	19221**
			SD	4295
			N	19
4	Ethylal	1000	Mean (pg/mL)	19879**
			SD	3982
			N	19

Excluding non-pregnant animals.

 

Table 11: Group mean values for TSH

Group	Treatment	mg/kg/day		Value
Statistical test				lWi
1	Control	0	Mean (pg/mL)	1342
			SD	479
			N	20
2	Ethylal	100	Mean (pg/mL)	1512
			SD	602
			N	20
3	Ethylal	330	Mean (pg/mL)	1294
			SD	432
			N	20
4	Ethylal	1000	Mean (pg/mL)	1945*
			SD	1087
			N	19

Excluding non-pregnant animals.

 

Table 12: Summary of litter data

Group	No. of pregnant females	No. of females with live young on Day 20	Pre-implantation loss (a)	Post-implantation (b)	Percent of live offspring to implantations (c)
1	20	20	46	15	95.4
2	20	20	33	11	96.6
3	20	20	47	23	92.2
4	20	19	31	21	95.5

a – Total pre-implantation loss considered to be, total number of corpora lutea – total number of implantations. Where implantations exceed corpora lutea, number of implantations will be considered to be equal to corpora lutea.

b – Total post-implantation loss is considered to be, total number of implantations – total number of live young.

c - Percentage of live young is considered to be = (100 – ‘post-implantation loss’)

 

Table 13: Litter data - group mean values on Day 20 of gestation

Group /Sex		Corpora lutea	Implantations	Resorptions			Implantation loss (%)		Live young			Sex ratio (%M)
				Early	Late	Total	Pre-	Post-	Male	Female	Total
Statistics test		Wi	Sh				Wi	Wi			Sh	Wi
1F	Mean	18.7	16.5	0.8	0.0	0.8	11.0	4.6	8.1	7.6	15.7	51.8
	SD	2.87	1.00	0.85	0.00	0.85	9.99	5.23	1.92	2.09	1.42	12.56
	N	20	20	20	20	20	20	20	20	20	20	20
2F	Mean	17.4	15.8	0.5	0.1	0.6	9.4	3.4	7.1	8.2	15.3	46.4
	SD	1.53	2.17	0.83	0.22	0.83	11.26	5.25	2.47	2.28	2.17	13.47
	N	20	20	20	20	20	20	20	20	20	20	20
3F	Mean	18.2	15.9	1.2	0.0	1.2	12.0	7.8	7.3	7.4	14.7	49.1
	SD	2.84	2.68	1.27	0.00	1.27	13.97	8.92	3.06	2.58	3.26	16.20
	N	20	20	20	20	20	20	20	20	20	20	20
4F	Mean	17.1	16.1	0.6	0.1	0.7	7.4	4.5	7.9	7.4	15.4	50.2
	SD	2.61	1.96	1.02	0.32	1.06	7.14	7.25	3.21	1.80	2.43	16.07
	N	19	19	19	19	19	19	19	19	19	19	19

 

Table 14: Placental, litter and fetal weights – group mean values (g) on Day 20 of gestation

Group /Sex		Placental weight	Total litter weight	Male fetal weight	Female fetal weight	Overall fetal weight
Statistics test		Wi	Wi	Wi	Wi	Wi
1F	Mean	0.53	58.66	3.86	3.63	3.74
	SD	0.085	5.922	0.221	0.204	0.212
	N	20	20	20	20	20
2F	Mean	0.54	57.32	3.88	3.67	3.77
	SD	0.065	8.618	0.273	0.302	0.285
	N	20	20	20	20	20
3F	Mean	0.54	55.37	3.86	3.67	3.78
	SD	0.060	12.055	0.281	0.223	0.220
	N	20	20	20	20	20
4F	Mean	0.52	56.40	3.78	3.58	3.67
	SD	0.067	8.924	0.212	0.207	0.196
	N	19	19	19	19	19

 

 

 

 

Table 15: Fetal examinations - major abnormality findings - group incidences

		Fetuses				Litters
Group		1	2	3	4	1	2	3	4
Number Examined		314	305	294	292	20	20	20	19
Total Number Affected		1	1	0	0	1	1	0	0
Cervical/Thoracic
Visceral	Retroesophageal right subclavian artery Dorsally displaced pulmonary trunk Muscular ventricular septal defect Malrotated heart	0 0 0 0	1 1 1 1	0 0 0 0	0 0 0 0	0 0 0 0	1 1 1 1	0 0 0 0	0 0 0 0
Appendicular
Skeletal	Thickened/misshapen long bones Polydactyly forepaw(s)	1 1	0 0	0 0	0 0	1 1	0 0	0 0	0 0

Note: individual fetuses/litters may occur in more than one category

Table 16: Fetal examinations – Major Historical Control Data

Species/strain/source: Rat/ Sprague Dawley/ Charles River UK

Necropsy date range: May 2018– February 2019

Number of studies: 8

Study types: Main

		TR83VY								HCD Range
		Fetuses				Litters
Group		1	2	3	4	1	2	3	4
Number Examined		314	305	294	292	20	20	20	19	2087	143
Cervical/Thoracic Visceral	Retroesophageal right subclavian artery Dorsally displaced pulmonary trunk Muscular ventricular septal defect Malrotated heart	0 0 0 0	1 1 1 1	0 0 0 0	0 0 0 0	0 0 0 0	1 1 1 1	0 0 0 0	0 0 0 0	0-1 0-0 0-0 0-0	0-1 0-0 0-0 0-0

 

 

Table 17: Fetal examinations – minor skeletal abnormality and variants findings – group incidences

		Fetuses				Litters
Group		1	2	3	4	1	2	3	4
Number Examined		158	153	146	146	20	20	20	19
Minor skeletal abnormalities
Cranial	sutural bone(s) interparietal fissure(s)	0 1	1 1	1 0	0 0	0 1	1 1	1 0	0 0
Vertebral element abnormality	thoracic	0	0	1	1	0	0	1	1
Ribs	medially thickened/kinked	1	0	0	0	1	0	0	0
Sternebrae	misaligned hemicentres	0	0	0	1	0	0	0	1
Costal cartilage	misaligned	0	0	0	1	0	0	0	1
Appendicular	misshapen cranial margin scapula(e)	0	0	1	3	0	0	1	1
Total affected by one or more of the above		2	2	3	5	2	2	3	3
Rib and vertebral configuration
Cervical rib	short supernumerary	2	1	0	0	1	1	0	0
13th rib	short without costal cartilage	1	2	0	1	1	1	0	1
Number of 14th ribs	short supernumerary full supernumerary total	5 0 5	5 0 5	6 0 6	11 2 13	5 0 5	3 0 3	4 0 4	5 1 5
Thoracolumbar vertebrae	18	0	0	1	0	0	0	1	0
Pelvic girdle	unilateral cranial shift	1	0	1	0	1	0	1	0

Note: individual fetuses/litters may occur in more than one category

Table 17 (cont): Fetal examinations – minor skeletal abnormality and variants findings – group incidences

		Fetuses				Litters
Group		1	2	3	4	1	2	3	4
Number Examined		158	153	146	146	20	20	20	19
Delayed/Incomplete ossification/unossified
Cranial	large nasofrontal suture cranial centres presphenoid hyoid	0 8 1 26	0 12 0 16	2 9 0 31	0 1 0 6	0 4 1 11	0 7 0 7	2 4 0 15	0 1 0 5
Sternebrae	5th and/or 6^th other total	98 10 99	76 3 78	60 5 61	70 7 73	18 9 18	18 1 18	19 3 19	18 3 18
Vertebrae	cervical thoracic sacrocaudal caudal	3 8 13 1	3 10 8 0	4 10 7 1	1 9 4 0	3 6 6 1	1 6 6 0	2 7 6 1	1 6 3 0
Ribs	any	0	0	1	0	0	0	1	0
Appendicular	pelvic bones metacarpals metatarsals	11 1 1	9 0 0	7 2 1	0 0 0	5 1 1	4 0 0	6 2 1	0 0 0
Increased ossification
Cervical vertebral centra	all ossified	0	0	1	1	0	0	1	1

Note: individual fetuses/litters may occur in more than one category

Table 18: Fetal examinations - Skeletal Historical Control Data

Species/strain/source: Rat/ Sprague Dawley/ Charles River UK

Necropsy date range: May 2018– February 2019

Number of studies: 8

Study types: Main

		TR83VY								HCD Range
		Fetuses				Litters
Group		1	2	3	4	1	2	3	4
Number Examined		158	153	146	146	20	20	20	19	1097	143
Minor skeletal abnormalities
Cranial Vertebral element abnormality Sternebrae Costal cartilage Appendicular	sutural bone(s) thoracic misaligned hemicentres misaligned misshapen cranial margin scapula(e)	0 0 0 0 0	1 0 0 0 0	1 1 0 0 1	0 1 1 1 3	0 0 0 0 0	1 0 0 0 0	1 1 0 0 1	0 1 1 1 1	0-2 0-3 0-2 0-2 0-1	0-1 0-2 0-2 0-2 0-1
Rib and vertebral configuration Number of 14th ribs Thoracolumbar vertebrae	full supernumerary 18	0 0	0 0	0 1	2 0	0 0	0 0	0 1	1 0	0-1 0-2	0-1 0-1
Delayed/Incomplete ossification/unossified Cranial Ribs Increased ossification Cervical vertebral centra	large nasofrontal suture any   all ossified	0 0   0	0 0   0	2 1   1	0 0   1	0 0   0	0 0   0	2 1   1	0 0   1	0-2 0-1   0-1	0-2 0-1   0-1

 

 

Table 19: Fetal examinations - minor visceral abnormality and necropsy findings - group incidences

		Fetuses				Litters
Group		1	2	3	4	1	2	3	4
Number Examined		156	152	148	146	20	20	20	19
Total Number Affected		25	23	21	11	15	16	13	9
Visceral abnormalities
Thymus	partially undescended lobe thymic remnant	3 1	2 0	4 0	1 0	3 1	1 0	4 0	1 0
Lungs	absent accessory lobe	0	1	0	0	0	1	0	0
Diaphragm	thinning with liver protrusion	1	0	0	0	1	0	0	0
Liver	fissured posterior caudate lobe folded posterior caudate lobe	1 0	0 0	0 1	1 0	1 0	0 0	0 1	1 0
Spleen	small	0	1	0	0	0	1	0	0
Testis(es)	malpositioned	1	1	1	0	1	1	1	0
Umbilical artery	left	2	2	1	0	2	2	1	0
Haemorrhages
Head	brain vitreous humour eye	1 2	2 0	2 0	2 0	1 2	2 0	2 0	2 0
Neck/Thorax	dorsal fat pad thoracic cavity	1 0	1 0	0 1	1 0	1 0	1 0	0 1	1 0
Abdomen	abdominal cavity liver lobes	10 2	9 3	10 1	5 2	7 2	6 3	8 1	4 2
General	subcutaneous	1	1	1	0	1	1	1	0
Necropsy observations (external)
Skin	shiny subcutaneous edema	0 0	2 1	2 0	0 0	0 0	2 1	2 0	0 0

Note: individual fetuses/litters may occur in more than one category

Table 20: Fetal examinations – Visceral Historical Control Data

Species/strain/source: Rat/ Sprague Dawley/ Charles River UK

Necropsy date range: May 2018– February 2019

Number of studies: 8

Study types: Main

		Fetuses				Litters				HCD Range
Group		1	2	3	4	1	2	3	4
Number Examined		156	152	148	146	20	20	20	19
Total Number Affected		25	23	21	11	15	16	13	9	1091	19
Visceral abnormalities Lungs   Spleen	absent accessory lobe folded posterior caudate lobe small	0 0 0	1 0 1	0 1 0	0 0 0	0 0 0	1 0 1	0 1 0	0 0 0	0-0 0-3 0-0	0-0 0-3 0-0
Haemorrhages	thoracic cavity	0	0	1	0	0	0	1	0	0-1	0-1
Necropsy observations (external) Skin	shiny subcutaneous edema	0 0	2 1	2 0	0 0	0 0	2 1	2 0	0 0	0-1 0-1	0-1 0-1

 

Table 21: Ano-genital distance - group mean absolute and adjusted values for fetuses

Group /Sex		Fetal weight (g)	Ano-genital distance (mm)
Statistics test		Wi
1M	Mean	3.9	4.1
	SD	0.22	0.19
	N	20	20
2M	Mean	3.9	4.0
	SD	0.28	0.21
	N	20	20
3M	Mean	3.9	4.1
	SD	0.28	0.20
	N	20	20
4M	Mean	3.8	4.1
	SD	0.21	0.14
	N	19	19

 

		Ano-genital distance (mm)
Statistics test		Wi
1M	Adjusted Mean	4.1
2M	Adjusted Mean	4.0
3M	Adjusted Mean	4.1
4M	Adjusted Mean	4.1

 

Table 21 (cont): Ano-genital distance - group mean absolute and adjusted values for fetuses

Group /Sex		Fetal weight (g)	Ano-genital distance (mm)
Statistics test		Wi
1F	Mean	3.6	2.5
	SD	0.20	0.13
	N	20	20
2F	Mean	3.7	2.5
	SD	0.26	0.18
	N	20	20
3F	Mean	3.7	2.5
	SD	0.23	0.20
	N	20	20
4F	Mean	3.6	2.6
	SD	0.21	0.21
	N	19	19

 

		Ano-genital distance (mm)
Statistics test		Wi
1F	Adjusted Mean	2.5
2F	Adjusted Mean	2.5
3F	Adjusted Mean	2.5
4F	Adjusted Mean	2.6

Table 22: Thyroid hormone data

Species/strain/source: Rat/ Sprague Dawley/ Charles River UK

Necropsy date range: Sept 2019– December 2019

Number of studies: 4

Study types: Main

	T3 (pg/mL)	T4 (pg/mL)	TSH (pg/mL)
N	80	80	80
1%	287	8204	303
5%	326	9916	419
50%	473	16950	1030
95%	625	26275	2476
99%	701	50765	2974
Mean	483	18730	1250
SD	97.8	13191.3	702.3

Conclusions:

Based on the results of this study, the maternal and embryo-fetal No-Observed-Adverse-Effect-Level (NOAEL) was 1000 mg/kg/day.

Executive summary:

Introduction

The purpose of this study was to assess the influence of Ethylal (an industrial chemical) on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Sprague Dawley rat.

Four groups of 20 females received Ethylal at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil at the same volume dose as treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined; macroscopic and microscopic pathology investigation were undertaken on Day 20 after mating and the gravid uterus weight and thyroid weight were recorded. Ano‑genital distance were measured for all fetuses and were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

Results

Following initial treatment (gestation day 6), signs associated with dosing Ethylal at 1000 mg/kg/day included unsteady gait in 11 animals, decreased activity in 5 animals, flattened posture, or gait, in 4 animals and partially closed eye lids in 3 animals. At 1000 mg/kg/day, chin rubbing was seen towards the end of treatment and at 330 mg/kg/day, or above, salivation was seen throughout the treatment period.

There was no clear difference in the overall (gestation day 6 to 20) body weight change for females treated with Ethylal at any dose level. Although body weight loss/stasis was seen in females receiving 330 or 1000 mg/kg/day following initial treatment gestation Day 6 to 7.

There was no effect of treatment on gravid uterine weight.

Food consumption of females treated at 1000 mg/kg/day was statistically significantly low prior to the start of treatment and remained low throughout treatment, although statistical significance was not attained on the last recording occasion.

There was no effect of treatment on the macroscopic appearance of organs in the adults or the external appearance of fetuses at necropsy.

The microscopic examination of the thyroid and parathyroid revealed no test item-related lesions.

There were no clear effects of treatment on early or late resorptions, numbers of live young, sex ratio or pre/post implantation losses.

There were no effects of treatment on placental, litter or fetal weights.

There was no effect of treatment on ano-genital distances. There was a slight dose related decrease in levels of T3 at 330 or 1000 mg/kg/day, a slight increase in T4 levels at 330 or 1000 mg/kg/day and a slight increase in TSH levels at 1000 mg/kg/day.

The incidence of major and minor abnormalities and skeletal variants in fetuses show no relationship to treatment.

Conclusion

Based on the results of this study, the maternal and the embryo-fetal No-Observed-Adverse-Effect-Level (NOAEL) was 1000 mg/kg/day.

Effect on developmental toxicity: 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:

Endpoint derived from an OECD TG 414 (GLP) study with ethylal. RSS of the test report is provided.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Ethylal is not classified in absence of adverse effects observed in 2 OECD 421 and OECD 414 (and both GLP) studies where NOAEL was 1000 mg/kg/day, corresponding to the highest tested concentration.