Diethoxymethane

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

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.

Test material

Test material information

Constituent 1

Reference substance name:

Diethoxymethane

EC Number:

207-330-6

EC Name:

Diethoxymethane

Cas Number:

462-95-3

Molecular formula:

C5H12O2

IUPAC Name:

diethoxymethane

Test material form:

liquid

Specific details on test material used for the study:

Colourless liquid.
Batch number: 1709141400R

Test animals

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.

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control - corn oil

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

600 mg/kg bw/day

Doses / concentrations 4

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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

Details on results (P0)

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

Effect levels (P0)

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

Target system / organ toxicity (P0)

1

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

2

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

female reproductive system

Organ:

ovary

uterus

Results: F1 generation

General toxicity (F1)

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.

Details on results (F1)

See tables 15-19

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

endocrine system

Organ:

thyroid gland

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

no

Any other information on results incl. tables

         

 

	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

Applicant's summary and conclusion

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.