Zinc bis(dibutyldithiocarbamate)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

This information will be updated when the final study report is available.

Effects on developmental toxicity

Description of key information

- Oral: NOAEL for maternal toxicity is 250 mg/kg bw/day; the NOAEL for developmental toxicity is 75 mg/kg bw/day, rat, OECD 414, 2018

- Oral: NOAEL for maternal toxicity and developmental toxicity are 3330 ppm (corresponding to an actual test item intake of 101 mg/kg/day), rabbit, OECD 414, 2021

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Oct 2020 to 22 Feb 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 18-19 weeks old
- Weight at study initiation: 2911 and 4086 g
- Housing: The animals were individually housed in cages with perforated floors equipped with water bottles
- Diet: Animals had free access to standard powder diet for rabbits, refreshed daily
- Water (e.g. ad libitum): Municipal tap water, ad libitum
- Acclimation period: At least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Targeted: 17 to 21, actual mean: 17 to 19°C
- Humidity (%): Targeted: 40 to 70, actual mean: 42 to 65
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 14 Oct 2020 to 22 Feb 2021

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. Standard powder diet for rabbits were used.

DIET PREPARATION
- Rate of preparation of diet: Diets were prepared at least once weekly for use at room temperature for a maximum of one day. Diets were kept in the freezer (≤-15°C) for a maximum of 8 days prior to use, if not used on the day of preparation. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of one day
- Storage temperature of food: Diets were kept in the freezer (≤-15°C) for a maximum of 8 days prior to use, if not used on the day of preparation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

See ''Any other information on material and methods''.

Details on mating procedure:

Day 0 post-coitum is the day of successful mating

Duration of treatment / exposure:

Day 7 to Day 29 post-coitum, inclusive.

Frequency of treatment:

Continuously

Duration of test:

29 days

Dose / conc.:

2 220 ppm

Remarks:

Corresponded with an intended dose level of approximately 66 mg/kg bw/day

Dose / conc.:

3 330 ppm

Remarks:

Corresponded with an intended dose level of approximately 100 mg/kg bw/day

Dose / conc.:

5 000 ppm

Remarks:

Corresponded with an intended dose level of approximately 150 mg/kg bw/day

No. of animals per sex per dose:

22

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: For the test substance, a pilot developmental toxicity study in rabbits was previously performed via oral with corn oil as a vehicle. Treatment of rabbits during the pilot developmental toxicity study resulted in low to absent food consumption, low food consumption and reduced faeces production across all groups. In another pilot developmental study in rabbits with a structural similar compound, using corn oil as vehicle, all animals, including controls, had to be sacrificed between post coitum Days 14 and 17 due to poor health conditions (low or absent food consumption, low body weights and reduced faeces production) and the study had to be terminated. Based on these findings it was concluded that pregnant rabbits housed and treated under the conditions of this study did not tolerate corn oil as vehicle. As no other suitable vehicle is available, it was concluded that pregnant rabbits housed and treated under the conditions of this study did not tolerate corn oil as vehicle when administered at a dose volume of 2 mL/kg. This was confirmed by a dedicated pilot study, in which the tolerability of corn oil (2 mL/kg) was tested after maternal exposure during the critical period of organogenesis when given orally by gavage in rabbits after optimisation of several husbandry conditions. Due to the physicochemical properties of this group of substances (hydrolytical instability), the use of aqueous vehicles was not an option. As no other suitable vehicle was available for administration in rabbits, it was decided in consultation with the Sponsor to perform the current pilot and main developmental toxicity study with the test substance in rabbits via dietary administration. administration. The dose levels were will be selected based on the results of the dose range finder, in consultation with the Sponsor and in an attempt to produce graded responses to the test substance.
Results of the DRF showed At 6700 and 10050 ppm, body weight gain was reduced during the entire treatment period and mean food consumption was (statistically significantly) decreased from the start of treatment onwards (Day 7-8 post-coitum). Based on the high incidence of early deliveries of females at 6700 ppm and 10050 ppm, which may result in an insufficient number of litters for evaluation of developmental data, 5000 ppm was selected as the highest dose level for the Main study

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily throughout the study.
- Cage side observations checked: Animals were observed for general health/mortality and moribundity. Animals will not be removed from the cage
during observation, unless necessary for identification or confirmation of possible findings.

CLINICAL OBSERVATIONS:
- Time schedule: From Day 7 post-coitum onwards up to the day prior to necropsy, animals were observed at least once daily.
- Animals were observed for specific clinical signs. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade were predefined at 1, 2, 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (i.e. maximum grade 1) were scored. Cage debris was examined to detect premature birth, if applicable.

BODY WEIGHT:
- Time schedule for examinations: Animals were weighed on Days 7, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Food consumption of animals was measured daily from Day 3 post-coitum onwards.

WATER CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: Regular basis throughout the study, by visual inspection.

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 29
- Organs examined: All animals were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs.

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

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions were analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. In case deemed appropriate, values were also be expressed as a percentage of predose or control values. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: group 2 vs group 1, group 3 vs group 1 and group 4 vs group 1.
Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution were compared using the Mann Whitney test. Mean litter proportions (percent per litter) of total fetal malformations and developmental
variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

Indices:

An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test was significant. No statistics was applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and postimplantation loss.

For each group, the following calculations were performed:
Preimplantation loss (%): (number of corpora lutea - number of implantation sites) / number of corpora lutea x 100
Postimplantation loss (%): (number of implantation sites - number of live foetuses) / number of implantation sites x 100

The foetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:
Viable foetuses affected/litter (%): number of viable fetuses affected/litter / number of viable fetuses/litter x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Reduced faeces production was observed in 13/22, 20/22, 20/22 and 20/22 animals of the control, 2220, 3330 and 5000 ppm groups, respectively, which corresponded with periods of reduced food consumption. In general, reduced faeces production was observed more
frequently and at a higher severity during the treatment period of animals treated at 5000 ppm. Female No. 66 treated at 3330 ppm was observed with piloerection and/or scored as lean between post-coitum Days 21-27. As this finding was incidental, occurred in the mid-dose
group only and was transient, it was considered not related to treatment with the test item. Other incidental findings noted during the treatment period in some animals among all groups included alopecia, scabs and/or piloerection. In addition, swelling, erythema, wound and white staining of the skin (throat region) were noted in Female No. 33 treated at 2220 ppm and alopecia on the last day of the study. These clinical signs occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test substance.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Before start of treatment on post-coitum Day 7, mean body weights were slightly lower compared to the initial body weight on post-coitum Day 0 in all groups, due to acclimatisation to the powder diet.
At 5000 ppm, body weight gain was reduced from post-coitum Day 15 onwards (7% vs 13% for concurrent controls on Day 29; not always statistically significant). The statistically significant lower body weight gain for the highest dose group from day 15 and onwards did
not result in a significant reduction in mean absolute body weight at the end of the treatment (-2%) due to higher absolute body weights (+3%) in the highest dose group compared to the control at the start of the treatment.
Mean body weight loss (corrected for the weight of gravid uterus) was observed in all groups, including controls. However, in females at 5000 ppm weight loss was more pronounced (-250.4 vs -104.8 gram in control (4.2% lower corrected weight gain in percent of weight on post-coitum Day 7 compared to control).
Body weights and (corrected) body weight gain at 2220 and 3330 ppm were considered unaffected by treatment with the test substance.
The statistically significant change in body weights on post-coitum Day 15 at 2220 ppm was considered to be unrelated to treatment with the test substance as no trend was apparent regarding dose.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During acclimatisation (post-coitum Days 3-7), mean food consumption was low for several females of all groups due to acclimatization to the powder diet. For most of these females, food consumption recovered to similar values as observed for other animals of the same dose group. Therefore, it was considered that the low food consumption at the end of the acclimatisation period did not affect the outcome of the study.
During treatment, mean food consumption (both absolute and relative) of females treated at 5000 ppm was statistically significantly lower than in controls from post-coitum Day 8 onwards (except for Day 15 to 17 for relative food consumption, and Day 15 for absolute food consumption). Overall, mean over mean (relative) food consumption over the postcoitum period at 5000 ppm was 19% lower than concurrent controls.
Food consumption tended to be slightly lower in females treated at 2220 and 3330 ppm between post-coitum Days 12-19, reaching statistical significance on several occasions at 2220 ppm only. Relative food consumption was decreased from post-coitum Days 12-15 (not always statistically significant), which recovered to values comparable to concurrent controls from post-coitum Days 16 onwards. Overall, mean over mean relative food consumption in the groups treated at 2220 and 3330 ppm over the post-coitum period was comparable to concurrent controls. As no trend was apparent regarding dose and duration of treatment, these minimal changes were considered unrelated to treatment with the test substance.

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations at scheduled necropsy did not reveal any alterations that were considered to have arisen as a result of treatment with the test substance.
At 5000 ppm, 3/22 females were observed with an enlarged gallbladder. As this finding occurred in a few animals only and was not accompanied by clinical observations or other macroscopic abnormalities of the gallbladder (i.e. obstruction), this finding was considered not related to treatment with the test substance.
Other findings that were noted among control and/or treated animals were considered to be of no toxicological significance, since they remained within the range of biological variation for rabbits of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

None of the animals aborted.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The mean percentage of pre-implantation loss was relatively high at 5000 ppm (13.5% vs 11.2% in concurrent controls) and the mean value was outside the range of the available historical control range. Notably, a relatively high percentage of pre-implantation loss was also noted in the concurrent controls, and treatment was initiated after implantation was completed. Therefore, this finding was considered unrelated to treatment with the test substance

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

The mean percentage of late resorptions was slightly increased at 5000 ppm (5.5% vs 2.3% in concurrent controls, although not statistically significant and within the range of historical control data) This resulted in a higher mean percentage of post-implantation loss (12.2% vs 6.5% in concurrent controls; not statistically significant and within the range of historical control data). The higher percentage of late resorptions at 5000 ppm was mainly attributed to two females. Female No. 80 was observed with 15 implantation sites of which 11 viable fetuses and 4 late resorptions, and Female No. 85 was observed with 16 implantation sites of which 10 viable fetuses and 6 late resorptions. As these changes were mainly attributed to these two females and values remained within the range of historical control data, this was considered not related to treatment with the test substance.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Three females in the control group, two females in the 2220 ppm group and one female in the 3330 ppm group were not pregnant at necropsy. The incidence of non-pregnancy was considered to be unrelated to treatment with the test item as no dose-related response was observed.

Other effects:

no effects observed

Description (incidence and severity):

The number of corpora lutea, implantation sites, viable and dead foetuses and early resorptions were similar and in the range of normal biological variation

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

3 330 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: Corresponding to an actual test substance intake of 101 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal developmental toxicity

Effect level:

>= 5 000 ppm

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Corresponding to a mean actual test item intake of 125 mg/kg bw/day.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean combined (male and female) foetal body weights were 41.6, 36.9, 37.2 and 36.2 gram for the control, 2220, 3330 and 5000 ppm groups, respectively.
Mean male, female and combined (male and female) foetal weights were lower at all dose levels compared to concurrent control (up to 12%, 11% and 15% at 2220, 3330 and 5000 ppm, respectively). Although mean fetal weights of the control group were relatively high compared to the historical control data, the mean male, female and combined (male and female) foetal body weights per litter at 5000 ppm were below the lower end of the 5th percentile of the historical control data, even including the foetal weights from Litter No. 82, with a relatively high mean fetal weight attributed to two fetuses only. Looking at the mean foetal weight per litter in this group, a mean litter weight below the range of historical control data was noted for 13/22 litters. At 2220 and 3330 ppm, mean fetal weight values remained within the range of historical control data, except for the male foetal weights at 2220 ppm.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to 5000 ppm.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

- There were no test substance-related effects on litter size up to 5000 ppm. The numbers of fetuses (litters) available for a full fetal morphological examination were 174 (19), 206 (20), 214 (21) and 215 (22) in the control, 2220, 3330 and 5000 ppm groups, respectively.
- Mean litter sizes were 9.2, 10.3, 10.2 and 9.8 fetuses/litter for the control, 2220, 3330 and 5000 ppm groups, respectively.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Of the foetuses from groups that received the test-substance two fetuses at the low dose were affected externally. One foetus had meningoencephalocele with matching skeletal abnormalities and an open eye, whereas fetus A039-2 had a distended abdomen with ascites possibly related to a large (right) atrium. As these abnormalities occurred at the low dose, they were considered chance findings.
Apart from a control foetus with omphalocele and a carpal flexure, whereby the radius was absent, no other malformations occurred and external variations were not seen in any group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were noted in 2 (2), 4 (3), 1 (1) and 1 (1) foetuses (litters) of the control, 2220, 3330 and 5000 ppm groups, respectively.
All skeletal malformations observed in the test-substance groups (vertebral anomaly with or without associated rib anomaly, caudal vertebral anomaly and sternal anomaly) were listed in our historical control data. Moreover, the incidental occurrence and/or group distribution does not indicate any relation to the test-substance
Among variations, (dose-related) signs of delays in ossification (unossified pubis, tarsals, metacarpals and/or metatarsals) were noted in all test-item groups and could be associated with the lower fetal weights observed in these groups. At the highest dose level the incidences of unossified tarsals (4.3%, not statistically significant) and unossified pubis (2.3%) were above the historical control maximum value and considered to be related to treatment with the test substance.
All other skeletal variations occurred at low incidences, in the absence of a dose-related incidence trend and/or at frequencies that were within the range of available historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral malformations occurred in 1 (1), 2 (2), 2 (1) and 1 (1) foetuses (litters) in the control, 2220, 3330 and 5000 ppm groups, respectively. As all malformations observed in test-substance groups occurred singly and were observed previously in historical control fetuses, they were considered chance findings.
The visceral variation ovary cyst showed a dose related increased incidence of 0.0%, 0.6%, 1.8% and 2.8% of fetuses per litter in the control, 2220, 3330 and 5000 ppm groups, respectively. The high dose incidence was above the historical control maximum value (2.0% per litter), and was considered related to treatment with the test substance.
All other visceral variations that were noted were considered unrelated to the test item as they occurred in the absence of a dose-related trend, infrequently and/or in control foetuses only.

Key result

Dose descriptor:

NOAEL

Effect level:

3 330 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Remarks on result:

other: Corresponding to an actual test item intake of 101 mg/kg bw/day

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Result diet analysis:

The concentrations analysed in the diets of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%). A small response at the retention time of the test item was observed in the chromatograms of the Group 1 diet. It was considered not to derive from the diet since a similar response was obtained in the matrix dissolved in 4% nitric acid in water. The diets of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Table 1. Body weights (summary in gram) females, F0 generation

		Group 1 Control	Group 2 2220 ppm	Group 3 3330 ppm	Group 4 5000 ppm
POSTCOITUM DAY 0	MEAN	3598	3517	3559	3671
	ST.DEV.	279.8	282.1	316.4	340.3
	N	19	20	21	22
DAY 7	MEAN	3452	3325	3391	3550
	ST.DEV.	243.2	246.8	226.1	265.2
	N	19	20	21	22
DAY 9	MEAN	3498	3353	3439	3595
	ST.DEV.	263.4	229.8	210.4	265.7
	N	19	20	21	22
DAY 12	MEAN	3554	3415	3483	3617
	ST.DEV.	231.8	242.7	214.0	276.2
	N	19	20	21	22
DAY 15	MEAN	3667	3467 *	3558	3675
	ST.DEV.	216.9	257.5	204.7	280.8
	N	19	20	21	22
DAY 18	MEAN	3693	3525	3587	3711
	ST.DEV.	207.3	254.0	194.1	284.3
	N	19	20	21	22
DAY 21	MEAN	3696	3539	3611	3706
	ST.DEV.	208.0	239.6	209.6	275.9
	N	19	20	21	22
DAY 24	MEAN	3747	3582	3655	3755
	ST.DEV.	213.2	247.9	212.8	284.5
	N	19	20	21	22
DAY 27	MEAN	3813	3630	3715	3788
	ST.DEV.	213.0	258.1	218.2	288.0
	N	19	20	21	22
DAY 29	MEAN	3883	3689	3768	3804
	ST.DEV.	218.7	270.1	213.7	300.8
	N	19	20	21	22

Explanations for excluded data are listed in the tables of the individual values. */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 2 Body weight gain (%) summary, F0 generation females

		Group 1 Control	Group 2 2220 ppm	Group 3 3330 ppm	Group 4 5000 ppm
DAY 7	MEAN	0	0	0	0
	ST.DEV.	0.0	0.0	0.0	0.0
	N	19	20	21	22
DAY 9	MEAN	1	1	1	1
	ST.DEV.	1.5	2.0	2.3	1.0
	N	19	20	21	22
DAY 12	MEAN	3	3	3	2
	ST.DEV.	2.0	2.1	3.4	1.7
	N	19	20	21	22
DAY 15	MEAN	6	4	5	4 *
	ST.DEV.	2.5	3.8	4.2	2.8
	N	19	20	21	22
DAY 18	MEAN	7	6	6	5
	ST.DEV.	3.0	3.7	4.6	2.9
	N	19	20	21	22
DAY 21	MEAN	7	7	7	4 *
	ST.DEV.	3.1	3.1	4.5	2.6
	N	19	20	21	22
DAY 24	MEAN	9	8	8	6 *
	ST.DEV.	3.1	3.6	5.1	3.0
	N	19	20	21	22
DAY 27	MEAN	11	9	10	7 *
	ST.DEV.	3.6	4.3	5.9	3.2
	N	19	20	21	22
DAY 29	MEAN	13	11	11	7 **
	ST.DEV.	3.9	4.6	6.4	3.3
	N	19	20	21	22

Explanations for excluded data are listed in the tables of the individual values. */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 3. Summary of maternal survival and pregnancy status

Dose group:	1		2		3		4
	NO.	%	NO.	%	NO.	%	NO.	%
Females on study	22		22		22		22
Females that aborted or deliverd	0	0	0	0	0	0	0	0
Females that died	0	0	0	0	0	0	0	0
Females that aborted	0	0	0	0	0	0	0	0
Nongravid	0	0	0	0	0	0	0	0
Gravid	0	0	0	0	0	0	0	0
Females that were euthanised	0	0	0	0	0	0	0	0
Nongravid	0	0	0	0	0	0	0	0
Gravid	0	0	0	0	0	0	0	0
Females examined at
Scheduled necropsy	22	100	22	100	22	100	22	100
Nongravid	3	13.6	2.0	9.1	1	5	0	0
Gravid	19	86.4	20.0	90.9	21.0	95.5	22	100
With resporptions only	0	0	0	0	0	0	0	0
With viable foetuses	19	100	20	100	21	100	22	100
Total females gravid	19	86.4	20.0	90.9	21.0	95.5	22	100

1- 0 ppm, 2- 2220 ppm, 3- 3330 ppm, 4- 5000 ppm

Table 4. Summary of foetal data at scheduled necropsy

Group		Sex (M)	Sex (F)	Viable foetuses	Dead foetuses	Resportion early	Resporption late	Post implantation loss	Implantation sites	Corpora lutea	Pre implantation loss	Foetal weights in grams	no. Of gravid females
1	TOTAL	83	91	174	0	9	5	14	188	213	25	NA	19
	MEAN	4.4	4.8	9.2	0.0	0.5	0.3	0.7	9.9	11.2	1.3	41.6
	S.D.	1.64	1.72	1.74	0.00	0.70	0.56	1.05	2.16	1.65	1.92	4.82
2	TOTAL	100	106	206	0	20	3	23	229	242	13	NA	20
	MEAN	5	5.3	10.3	0.0	1.0	0.2	1.2	11.5	12.1	0.7	36.9**
	S.D.	2.38	2.54	1.78	0.00	1.41	0.67	1.66	2.14	2.10	0.81	4.31
3	TOTAL	109	105	214	1	9	2	12	226	250	24	NA	21
	MEAN	5.2	5.0	10.2	0.0	0.4	0.1	0.6	10.8	11.9	1.1	37.2*
	S.D.	2.06	1.84	1.97	0.22	0.75	0.30	0.75	2.10	1.55	1.31	3.86
4	TOTAL	117	98	215	0	14	17	31	246	282	36	NA	22
	MEAN	5.3	4.5	9.8	0.0	0.6	0.8	1.4	11.2	12.8	1.6	36.2**
	S.D.	2.83	1.97	2.58	0.00	0.90	1.51	1.50	3.03	2.54	1.76	5.47

* = Significantly different from the control group at 0.05

** = Significantly different from the control group at 0.01

NA = NOT APPLICABLE

MEAN NUMBER OF VIABLE FETUSES, MEAN NUMBER OF IMPLANTATION SITES, MEAN NUMBER OF CORPORA LUTEA,

FOETAL WEIGHTS COMPARED USING DUNNETT'S TEST

1- 0 ppm, 2- 2220 ppm, 3- 3330 ppm, 4- 5000 ppm

Conclusions:

In conclusion, the maternal NOAEL is set at 3330 ppm (corresponding to an actual test item intake of 101 mg/kg/day), based on the decreased body weight gain and food consumption. The developmental NOAEL is set at 3330 ppm (corresponding to an actual test item intake of 101 mg/kg/day), based on the decreased mean foetal weights at 5000 ppm. A higher dose level than 5000 ppm was considered not feasible as a dose level of 6700 ppm resulted in a high incidence of early deliveries during the Dose Range Finder study

Executive summary:

The objectives of this study were to determine the potential of the test substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given via diet to time-mated female New Zealand White rabbits from Days 7 to 29 post-coitum, inclusive according to OECD TG 414 and GLP principles. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels in this study were selected to be 0, 2220, 3330, 5000 ppm (corresponding to a mean actual test item intake of 65, 101 and 125 mg/kg/day, respectively), based on the results of the Dose Range Finder. Chemical analyses of dietary preparations were conducted once during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, test item intake, macroscopic examination, organ weights, uterine contents, corpora lutea, implantation sites, and pre- and post-implantation loss. In addition, the following parameters were determined for the F1-generation: the number of live and dead foetuses, foetal body weights, sex ratio, external, visceral and skeletal malformations and developmental variations.

Results showed for maternal toxicity that no mortality occurred during the study period. At 5000 ppm (corresponding to an actual test item intake of 125 mg/kg/day), a lower mean food consumption (absolute and relative) was observed from post-coitum Day 7 onwards. This was accompanied with a reduced body weight gain during the entire treatment period and an increased incidence and severity of reduced faeces production. Additionally, body weight loss corrected for gravid uterus was lower compared to control (-250.4vs-104.8 gram in controls; 4.2% lower corrected weight gain in percent of weight on post-coitum Day 7 compared to control). As a dose-dependent reduced body weight gain and decreased food consumption were also noted in the Dose Range Finder at actual test item intake levels of 151 mg/kg/day (6700 ppm; intended dose level of 300 mg/kg/day) and 161 mg/kg/day (10050 ppm; intended dose level of 600 mg/kg/day), together with early deliveries of 3/6 females at 151 and 4/6 females at 161 mg/kg/day, these effects were considered to have a very steep dose-response relation. For this reason, although minor effects on body weight and food consumption were noted in the Main study, these effects cannot be neglected and were considered to be adverse. No test item-related gross findings were observed at necropsy. The number of corpora lutea, implantation sites, viable or dead foetuses, early or late resorptions, pre- and post-implantation loss were considered not affected by treatment with the test item up to 5000 ppm. For developmental toxicity, mean male, female and combined (male and female) foetal weights were lower in all treatment groups at 2220, 3330 and 5000 ppm when compared with controls. As mean foetal weights (per litter) at 5000 ppm were below the range of the historical control data, this decrease was considered to be adverse at this concentration. In addition, higher litter incidences of the skeletal variations unossified tarsals and unossified pubis were noted at 5000 ppm. Although statistical significance was only reached for the increased incidence of unossified pubis at 5000 ppm, the higher litter incidences of both unossified tarsals and unossified pubis at this concentration were outside the range of historical control data. Based on the individual lower foetal weights of most affected foetuses, this indication of a delayed ossification in most foetuses was considered a foetal weight effect and not a direct toxicological effect of the test substance. A dose related increase in mean litter incidence of ovary cysts was observed in all dose groups, with values above the historical control range at 5000 ppm, although not statistically significant. However, as this concerns a variation with no effect on development, it was considered to be non-adverse. No test item-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, malformations and external developmental variations).

In conclusion, the maternal NOAEL is set at 3330 ppm (corresponding to an actual test item intake of 101 mg/kg/day), based on the decreased body weight gain and food consumption. The developmental NOAEL is set at 3330 ppm (corresponding to an actual test substance intake of 101 mg/kg/day), based on the decreased mean foetal weights at 5000 ppm. A higher dose level than 5000 ppm was considered not feasible as a dose level of 6700 ppm resulted in a high incidence of early deliveries during the Dose Range Finder study

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Aug 2018 to 13 Sep 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 January 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 11-15 weeks
- Weight at study initiation: 177 – 262 g
- Housing: individually in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) provided ad libitum throughout the study
- Water: Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 49 to 79
- Air changes (per hr): ≥ 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
26 Aug 2018 to 13 Sep 2018

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The test item dosing formulations were mixed using the Ultra-Turrax. The dosing formulations were prepared daily as a suspension and dosed within 6 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: The use of corn oil was required due to the physicochemical properties of the test substance (low water solubility and stability)
- Concentration in vehicle: 6.25, 18.75 and 62.50 mg/mL
- Amount of vehicle: 4 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

CONCENTRATION ANALYSIS
Duplicate sets of samples (approximately 500 mg, accurately weighed) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration.

HOMOGENEITY ANALYSIS
Duplicate sets of samples (approximately 500 mg, accurately weighed) were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was =< 10%.

STABILITY ANALYSIS
As requested by the Sponsor, stability analysis of the test item in the vehicle was not determined since the available analytical method was only capable of measuring the zinc content in the test substance and, therefore, unable to measure the test item itself.

Details on mating procedure:

The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating).

Duration of treatment / exposure:

The test item and vehicle were administered 7 days a week from day 6 to day 20 post-coitum, inclusive.

Frequency of treatment:

Once daily

Duration of test:

Animals surviving until scheduled euthanasia were euthanised on day 21 post-coitum.

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

Group 1 to 4: 22 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of the range finder in the rat and in an attempt to produce graded responses to the test item. In this dose range finder, a single female treated with 500 mg/kg bw/day was sacrificed in extremis in a poor condition: body weight loss of 9% (Day 12 PC) and the following clinical signs were observed: a lean appearance, hunched posture, piloerection and ptosis. No macroscopic findings were noted at necropsy. For females treated at 500 mg/kg bw/day overall body weight loss and a reduction in food consumption were observed up to Day 12 PC. During Days 15-21 PC, the body weight gain remained reduced when compared with concurrent controls. Based on the corrected body weight gain of the dams, females at 500 mg/kg lost weight compared to concurrent controls which gained weight. Treatment related clinical signs such as hunched posture and piloerection were observed. At 250 mg/kg, slight body weight loss and a decreased food consumption was observed at Day 9 PC. Body weight and food consumption recovered during the remaining treatment period. The corrected body weight gain of the dams was slightly reduced when compared to concurrent controls. Piloerection and hunched posture were observed in most females during treatment. At 100 mg/kg, the body weight gain was slightly reduced when compared to concurrent control during treatment. The decrease in body weight gain showed no dose-related response, as the body weight gain of females at 250 mg/kg was less reduced than the females treated at 100 mg/kg. Food consumption was slightly reduced on Days 6-12 PC, but from Day 12 onwards the food consumption increased to normal values again. The corrected body weight of the dams was slightly reduced when compared to concurrent controls. Two out of 6 females presented with piloerection. No treatment-related changes were noted in the liver and kidney weights. Macroscopic observations at necropsy did not reveal any alterations that were considered to be toxicologically relevant. All females surviving up to scheduled necropsy were found to be pregnant with viable fetuses. No treatment related changes for the number of post-implantation loss were observed. Fetal findings Litter sizes were within normal limits for all groups. The male:female ratios were equal in litters of all groups. Combined fetal body weights at 250 and 500 mg/kg were notably lower compared with the concurrent control. External examination of the fetuses did not show any treatment related abnormalities.
Based on the results of the dose range finder, selected dose levels for the main study were 25, 100 and 250 mg/kg bw/day.

- Rationale for animal assignment: random

Maternal examinations:

CAGE SIDE OBSERVATIONS
- Time schedule: twice daily, in the morning and at the end of the working day
- Cage side observations included general health/mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: once daily, beginning on Day 2 post-coitum and lasting up to the day prior to necropsy.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.
Cage debris was examined to detect premature birth.

BODY WEIGHT
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION
- Time schedule for examinations: Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum
- Food consumption was determined for each animal and mean daily diet consumption calculated as g food/kg body weight/day

POST-MORTEM EXAMINATIONS
- Sacrifice on gestation day 21 post-coitum
- Organs examined: All animals were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). The liver and kidney were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organ to body weight ratio (using the body weight on Day 21 post-coitum) were calculated.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination
Examinations included:
- Gravid uterus weight
- Number of corpora lutea
- Number of implantations
- Number of early resorptions
- Number of late resorptions
- Other: the number and distribution of live and dead fetuses; the sex of each fetus based on the ano-genital distance

Fetal examinations:

- External examinations: all viable fetuses per litter. Each viable foetus was sexed, examined in detail to detect macroscopic visible abnormalities and their weight. A nonviable fetus (Group 4) was examined and weighed (the degree of autolysis was minimal or absent). Late resorptions with malformations were fixed in 10% buffered formalin.
- Soft tissue examinations: the sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development.
- Skeletal examinations: half per litter. As skeletal malformations were suspected for a ngle fetus selected for visceral examination, this fetus was also subjected to skeletal examination.
- Head examinations: half per litter. The heads were removed from half of the fetuses in each litter. This examination included serial sectioning of the head in at least seven even slices after removal of the lower jaw. For each slice, the front- and back side was examined. Multiple structures were distinguished, special attention was paid to nose/throat, eyes and brains.

Statistics:

STATISTICAL ANALYSIS
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analysed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

PARAMETRIC
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

NON-PARAMETRIC
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences.

INCIDENCE
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

MATERNAL VARIABLES
Body Weight Gains: Calculated against the body weight on Day 6 post-coitum
Corrected Body Weight Gains: Body weight on Day 21 post-coitum minus the body weight on Day 6 post-coitum and the weight of gravid uterus.
Relative Food Consumption: Calculated against the body weight for scheduled intervals.
Organ Weight Relative to Body Weight: Calculated against the body weight on Day 21 post-coitum

REPRODUCTION AND DEVELOPMENTAL VARIABLES
For each group, the following calculations were performed:

Pre-implantation loss (%): [(number of corpora lutea - number of implantation sites) / number of corpora lutea] x 100

Post-implantation loss (%)
[(number of implantation sites - number of live fetuses) / number of implantation sites] x 100

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:

Viable fetuses affected/litter (%): [(number of viable fetuses affected/litter) / (number of viable fetuses/litter)] x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Piloerection was noted in one, two and four females in the control, 75 and 250 mg/kg groups, respectively. In addition, two and four females at 250 mg/kg showed pale faeces and salivation, respectively. These symptoms were noted mostly for 2 to 10 consecutive days between Days 6-15 of treatment. In most females the clinical signs were transient, therefore these clinical findings were regarded as non-adverse.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain of females at 75 and 250 mg/kg bw/day was statistically significantly reduced throughout the treatment period when compared with concurrent control. This resulted in a decrease of 4 and 9% in mean body weight at 75 and 250 mg/kg bw/day, respectively, when compared with control on Day 21 post-coitum (statistically significant at 250 mg/kg bw/day only). Mean body weight of females at 25 mg/kg bw/day were also slightly lower when compared with concurrent control, but no statistical significance was achieved and was it therefore considered not toxicologically relevant.
The corrected body weight gain of the dams was reduced in a dose related trend from females at 25 mg/kg bw/day onwards; the decrease was statistically significant at 75 and 250 mg/kg bw/day when compared with concurrent control (6.3 and 2.9% vs. 14.0%, respectively). The mean corrected body weight gain of females at 75 and 250 mg/kg bw/day was below the P5-value of historical control data (P5 = 7.1 - 20.8%).
The correction for gravid uterus weight made clear that two pregnant females at 75 mg/kg bw/day and five pregnant females at 250 mg/kg bw/day actually lost weight over the treatment phase, i.e. Day 6 to Day 21 post-coitum.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 250 mg/kg bw/day, mean food consumption was decreased throughout the treatment period when compared to concurrent control. Overall, relative food consumption was decreased with 9% when compared with concurrent control (mean of means). The differences between females at 250 mg/kg bw/day and concurrent control ranged from 3-18% on separate intervals. Statistical significance was achieved during the first two intervals and the last one.
At 75 mg/kg, mean food consumption before or after allowance for body weight was statistically significantly reduced with 14% on Days 6-9 post-coitum when compared with concurrent control mean. This was followed by recovery in subsequent intervals.
On Days 18-20, relative food consumption was also slightly decreased with 6%, which was attributed to the extremely low food consumption of one female (5 gram/day). Food consumption of other females at 75 mg/kg bw/day remained similar to control. Therefore, the decrease in food consumption on Days 18-21 was considered incidental.
At 25 mg/kg, food consumption before and after correction for body weight remained in the same range as controls over the study period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ weights and organ:body weight ratios of treated animals were considered to be similar to those of control animals.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

The litter incidence of early and late resorptions was considered unaffected by treatment up to 250 mg/kg.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

effects observed, non-treatment-related

Description (incidence and severity):

There was one female at 250 mg/kg bw/day that delivered offspring on the day of scheduled necropsy (9 viable fetuses). At the low incidence observed (one high dose female only), this was considered to be unrelated to treatment.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The numbers of pregnant females in the control and test groups were similar.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal developmental

Effect level:

>= 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean male, female and combined fetal body weights were statistically significantly decreased with 7-8% at 250 mg/kg bw/day when compared with concurrent controls. Mean weights were just below the P5-value of historical control data (P5 is 5.2, 4.9 and 5.0 grams for male, female and combined mean fetal body weight, respectively).
The mean fetal body weights at 25 and 75 mg/kg bw/day remained in the same range as controls.
Mean combined (male and female) fetal body weights were 5.3, 5.3, 5.2 and 4.9 grams for the control, 25, 75 and 250 mg/kg groups, respectively.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to 250 mg/kg.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on litter size of any group.
The numbers of fetuses (litters) available for morphological examination were 245 (22), 233 (22), 260 (22) and 240 (21) in the control, 25, 75 and 250 mg/kg groups, respectively.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on external morphology following treatment up to 250 mg/kg bw/day.
The only external malformations were observed in the litter of one dam (25 mg/kg bw/day). One fetus in this litter had an absent tail (confirmed skeletally) and anal atresia and the late resorption in this litter had generalized oedema and a short tail. Because these malformations occurred in a single litter at the low dose level, they were considered to be of spontaneous origin.
External variations were not observed in any of the treatment groups.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on skeletal morphology following treatment up to 250 mg/kg bw/day.
Skeletal malformations were observed in one 25 mg/kg bw/day fetus and two control fetuses. Aside from the underlying skeletal malformation due to absence of the tail in one fetus at 25 mg/kg bw/day, this fetus appeared to have bent limb bones and a vertebral centra anomaly as well. Bent limb bones were also observed in one control fetus. The other affected control fetus had a vertebral anomaly with associated rib anomaly. Due to the single occurrence and/or occurrence in control fetuses these malformations were considered unrelated to treatment.
Skeletal variations occurred at an incidence of 76.2%, 65.6%, 78.9% and 60.6% per litter in the control, 25, 75 and 250 mg/kg bw/day groups, respectively. The incidence at the high dose level was statistically significantly lower than the control value, which was caused by two findings: bent ribs and reduced ossification of the skull. These respective findings occurred at an incidence of 7.8% and 9.3% in the control group, whereas both did not occur in the 250 mg/kg bw/day group. The reason for the absence of these variations at 250 mg/kg bw/day is unknown, but is considered not to have any detrimental effect or toxicological relevance.
All other variations occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within the range of available historical control data. Therefore, they were considered unrelated to treatment.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on visceral morphology following treatment up to 250 mg/kg bw/day. No treatment-related changes were noted in the heads which were removed from one-half of the fetuses in each litter.
Two viscerally malformed fetuses were observed. Besides a fetus at 75 mg/kg bw/day that had situs inversus, there was a control fetus for which internal hydrocephaly was observed during cephalic examination. Due to the single occurrence and/or occurrence in a control fetus both malformations were considered to have occurred by chance.
One visceral variation (small supernumerary liver lobes) was observed in one control fetus, two fetuses at 75 mg/kg bw/day (separate litters) and five fetuses at 250 mg/kg bw/day (separate litters), with a litter incidence of 0.8, 1.5 and 3.8%, respectively. The incidences remained well within the range of available historical control data and therefore the increase in incidence was considered unrelated to treatment.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

DOSE FORMULATION ANALYSIS

Accuracy

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). A small response at m/z 66 (isotope of quantification of the test item) was detected in the Group 1 formulation prepared for use in Week 1. It was not considered to derive from the formulation since a similar response was obtained in the analytical blanks. The maximum contribution to the samples at the lowest concentration level was 0.13%. It was considered that it has no significant effect on the results of the test samples.

Homogeneity

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

TABLE 1.6 BODY WEIGHTS (GRAM) SUMMARY FEMALES
F0-GENERATION
		GROUP 1	GROUP 2	GROUP 3	GROUP 4
		CONTROL	25 MG/KG	75 MG/KG	250 MG/KG
POST COITUM
DAY 2	MEAN	204	203	204	202
	ST.DEV.	11.8	16.7	15.4	17.4
	N	22	22	22	22
DAY 6	MEAN	221	221	223	220
	ST.DEV.	13	19.8	16.9	18.4
	N	22	22	22	22
DAY 9	MEAN	232	228	226	221
	ST.DEV.	13.7	18.2	16.5	17.5
	N	22	22	22	22
DAY 12	MEAN	248	245	242	232 **
	ST.DEV.	15.1	21.3	19	17.8
	N	22	22	22	22
DAY 15	MEAN	263	258	257	246 *
	ST.DEV.	17.6	22.5	20.3	17.3
	N	22	22	22	22
DAY 18	MEAN	294	289	289	276 *
	ST.DEV.	20.2	26	20.8	19.6
	N	22	22	22	22
DAY 21	MEAN	331	323	318	300 **
	ST.DEV.	26.6	32.1	26.7	28.2
	N	22	22	22	22
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

TABLE 1.7 BODY WEIGHT GAIN (%) SUMMARY FEMALES
F0-GENERATION
		GROUP 1	GROUP 2	GROUP 3	GROUP 4
		CONTROL	25 MG/KG	75 MG/KG	250 MG/KG
POST COITUM
DAY 2	MEAN	-8	-8	-8	-8
	ST.DEV.	1.5	1.5	1.8	1.6
	N	22	22	22	22
DAY 6	MEAN	0	0	0	0
	ST.DEV.	0	0	0	0
	N	22	22	22	22
DAY 9	MEAN	5	3	2 **	1 **
	ST.DEV.	2.1	2.6	2.1	2.6
	N	22	22	22	22
DAY 12	MEAN	12	11	8 **	5 **
	ST.DEV.	2.4	3.2	2.1	3.2
	N	22	22	22	22
DAY 15	MEAN	19	17	15 **	12 **
	ST.DEV.	2.8	3.8	2.9	4.1
	N	22	22	22	22
DAY 18	MEAN	33	31	29 *	26 **
	ST.DEV.	4.5	4.8	3.5	5.3
	N	22	22	22	22
DAY 21	MEAN	50	46	43 **	36 **
	ST.DEV.	6.8	6.7	8.3	7.9
	N	22	22	22	22
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

TABLE 1.8 FOOD CONSUMPTION (G/ANIMAL/DAY) SUMMARY FEMALES
F0-GENERATION
		GROUP 1	GROUP 2	GROUP 3	GROUP 4
		CONTROL	25 MG/KG	75 MG/KG	250 MG/KG
POST COITUM
DAYS 2-6	MEAN	23	22	23	22
	ST.DEV.	2.4	2.2	2.6	1.9
	N	22	22	22	22
DAYS 6-9	MEAN	19	18	16 **	15 **
	ST.DEV.	2.1	2.4	3.2	3.1
	N	22	22	22	22
DAYS 9-12	MEAN	21	21	19	16 **
	ST.DEV.	2.9	3.1	3.5	3.8
	N	22	22	22	22
DAYS 12-15	MEAN	18	19	18	17
	ST.DEV.	2.8	2.6	3	2.2
	N	22	22	22	22
DAYS 15-18	MEAN	24	23	23	21 **
	ST.DEV.	2.8	3.1	2.2	2.5
	N	22	22	22	22
DAYS 18-21	MEAN	23	22	21	18 **
	ST.DEV.	3.2	2.9	4.3	4.7
	N	21	22	22	21
MEAN OF MEANS		21	21	20	18
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

TABLE 1.9 RELATIVE FOOD CONSUMPTION (G/KG BODY WEIGHT/DAY) SUMMARY FEMALES
F0-GENERATION
		GROUP 1	GROUP 2	GROUP 3	GROUP 4
		CONTROL	25 MG/KG	75 MG/KG	250 MG/KG
POST COITUM
DAYS 2-6	MEAN	102	100	105	102
	ST.DEV.	7.2	7.5	10.4	7.9
	N	22	22	22	22
DAYS 6-9	MEAN	83	81	71 **	69 **
	ST.DEV.	6.6	9.6	13	15
	N	22	22	22	22
DAYS 9-12	MEAN	84	84	80	69 **
	ST.DEV.	8.5	9.3	12.1	13.9
	N	22	22	22	22
DAYS 12-15	MEAN	70	72	70	68
	ST.DEV.	7.7	7.1	8.8	7.9
	N	22	22	22	22
DAYS 15-18	MEAN	82	81	81	77
	ST.DEV.	7.3	9.9	6.3	7.7
	N	22	22	22	22
DAYS 18-21	MEAN	69	69	65	60 *
	ST.DEV.	5.9	7.2	12.1	12
	N	21	22	22	21
MEAN OF MEANS		81	81	78	74
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

TABLE 1.10 MACROSCOPIC FINDINGS SUMMARY FEMALES
	GROUP 1 CONTROL	GROUP 2	GROUP 3	GROUP 4
		25 MG/KG	75 MG/KG	250 MG/KG
END OF TREATMENT
Animals examined	22	22	22	22
Animals without findings	22	21	22	21
Animals affected	0	1	0	1
General observations	0	0	0	1
Early delivery
Liver	0	1	0	0
Diaphragmatic hernia
# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

TABLE 1.11 ORGAN WEIGHTS (GRAM) SUMMARY FEMALES
		GROUP 1	GROUP 2	GROUP 3	GROUP 4
		CONTROL	25 MG/KG	75 MG/KG	250 MG/KG
END OF TREATMENT
BODY W.	MEAN	331	323	318	300 **
(GRAM)	ST.DEV	27	32	27	28
	N	22	22	22	22
LIVER	MEAN	13.89	13.66	13.25	12.67
(GRAM)	ST.DEV	1.31	1.97	1.6	1.87
	N	22	22	22	22
KIDNEYS	MEAN	1.64	1.63	1.64	1.58
(GRAM)	ST.DEV	0.15	0.15	0.14	0.18
	N	22	22	22	22
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

TABLE 1.11 ORGAN/BODY WEIGHT RATIOS (%) SUMMARY FEMALES
		GROUP 1	GROUP 2	GROUP 3	GROUP 4
		CONTROL	25 MG/KG	75 MG/KG	250 MG/KG
END OF TREATMENT
BODY W.	MEAN	331	323	318	300 **
(GRAM)	ST.DEV	27	32	27	28
	N	22	22	22	22
LIVER	MEAN	4.2	4.23	4.16	4.22
(%)	ST.DEV	0.31	0.41	0.31	0.37
	N	22	22	22	22
KIDNEYS	MEAN	0.5	0.51	0.52	0.53
(%)	ST.DEV	0.04	0.03	0.06	0.03
	N	22	22	22	22
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

TABLE 1.12 SUMMARY OF MATERNAL SURVIVAL AND PREGNANCY STATUS
DOSE GROUP :	1		2		3		4
	NO.	%	NO.	%	NO.	%	NO.	%
FEMALES ON STUDY	22		22		22		22
FEMALES THAT ABORTED
OR DELIVERED	0	0	0	0	0	0	1	4.5
FEMALES THAT DIED	0	0	0	0	0	0	0	0
FEMALES THAT ABORTED	0	0	0	0	0	0	0	0
NONGRAVID	0	0	0	0	0	0	0	0
GRAVID	0	0	0	0	0	0	0	0
FEMALES THAT WERE EUTHANIZED	0	0	0	0	0	0	0	0
NONGRAVID	0	0	0	0	0	0	0	0
GRAVID	0	0	0	0	0	0	0	0
FEMALES EXAMINED AT
SCHEDULED NECROPSY	22	100	22	100	22	100	21	95.5
NONGRAVID	0	0	0	0	0	0	0	0
GRAVID	22	100	22	100	22	100	21	100
WITH RESORPTIONS ONLY	0	0	0	0	0	0	0	0
WITH VIABLE FETUSES	22	100	22	100	22	100	21	100
TOTAL FEMALES GRAVID	22	100	22	100	22	100	22	100
1- 0 MG/KG; 2- 25 MG/KG; 3- 75 MG/KG; 4- 250 MG/KG

TABLE 1.13 SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY
								POST			PRE	FETAL	NO. OF
SEX	VIABLE	SEX		VIABLE	DEAD	RESORPTIONS		IMPLANTATION	IMPLANTATION	CORPORA	IMPLANTATION	WEIGHTS	GRAVID
GROUP		M	F	FETUSES	FETUSES	EARLY	LATE	LOSS	SITES	LUTEA	LOSS	IN GRAMS	FEMALES
1	TOTAL	125	120	245	0	10	0	10	255	264	9	NA	22
	MEAN	5.7	5.5	11.1	0	0.5	0	0.5	11.6	12	0.4	5.3
	S.D.	1.78	2.36	1.58	0	0.8	0	0.8	1.53	1.54	0.8	0.23
2	TOTAL	124	109	233	0	13	1	14	247	265	18	NA	22
	MEAN	5.6	5	10.6	0	0.6	0	0.6	11.2	12	0.8	5.3
	S.D.	1.92	1.86	2.24	0	1.01	0.21	1.09	1.93	1.73	1.26	0.24
3	TOTAL	124	136	260	0	7	0	7	267	275	8	NA	22
	MEAN	5.6	6.2	11.8	0	0.3	0	0.3	12.1	12.5	0.4	5.2
	S.D.	1.81	1.37	1.53	0	0.65	0	0.65	1.32	1.1	0.66	0.45
4	TOTAL	124	116	240	1	9	1	11	251	256	5	NA	21
	MEAN	5.9	5.5	11.4	0	0.4	0	0.5	12	12.2	0.2	4.9**
	S.D.	1.81	2.44	1.66	0.22	0.6	0.22	0.68	1.47	1.29	0.54	0.32
** = Significantly different from the control group at 0.01
NA = NOT APPLICABLE
MEAN NUMBER OF VIABLE FETUSES, MEAN NUMBER OF IMPLANTATION SITES, MEAN NUMBER OF CORPORA LUTEA,
FETAL WEIGHTS COMPARED USING DUNNETT'S TEST
1- 0 MG/KG, 2- 25 MG/KG, 3- 75 MG/KG, 4- 250 MG/KG

TABLE 1.14 SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY [% PER LITTER]
GROUP:	0 MG/KG	25 MG/KG	75 MG/KG	250 MG/KG
CORPORA LUTEA
MEAN	12	12	12.5	12.2
S.D.	1.54	1.73	1.1	1.29
N	22	22	22	21
IMPLANTATION SITES
MEAN	11.6	11.2	12.1	12
S.D.	1.53	1.93	1.32	1.47
N	22	22	22	21
VIABLE FETUSES (%)
MEAN	96.2	94.1	97.3	95.5
S.D.	6.85	10.2	5.46	6.07
N	22	22	22	21
DEAD FETUSES (%)
MEAN	0	0	0	0.5
S.D.	0	0	0	2.18
N	22	22	22	21
EARLY RESORPTIONS (%)
MEAN	3.8	5.4	2.7	3.6
S.D.	6.85	9.25	5.46	5.1
N	22	22	22	21
LATE RESORPTIONS (%)
MEAN	0	0.5	0	0.4
S.D.	0	2.13	0	1.68
N	22	22	22	21
TOTAL RESORPTIONS (%)
MEAN	3.8	5.9	2.7	4
S.D.	6.85	10.2	5.46	5.1
N	22	22	22	21
PRE-IMPLANTATION LOSS (%)
MEAN	3.3	6.5	2.9	2
S.D.	6.13	10.23	5.41	4.55
N	22	22	22	21
POST-IMPLANTATION LOSS (%)
MEAN	3.8	5.9	2.7	4.5
S.D.	6.85	10.2	5.46	6.07
N	22	22	22	21
MALES (%)
MEAN	52.1	53.5	47.2	52.5
S.D.	17.41	14.83	12.2	16.26
N	22	22	22	21
FEMALES (%)
MEAN	47.9	46.5	52.8	47.5
S.D.	17.41	14.83	12.2	16.26
N	22	22	22	21
MALE FETAL WEIGHTS (g)
MEAN	5.4	5.4	5.3	5.0**
S.D.	0.24	0.23	0.5	0.34
N	22	22	22	21
FEMALE FETAL WEIGHTS (g)
MEAN	5.2	5.1	5.1	4.8**
S.D.	0.24	0.25	0.46	0.36
N	22	22	22	21
COMBINED FETAL WEIGHTS (g)
MEAN	5.3	5.3	5.2	4.9**
S.D.	0.23	0.24	0.45	0.32
N	22	22	22	21
PROPORTIONAL (%) DATA COMPARED USING THE MANN-WHITNEY TEST
FETAL WEIGHTS, CORPORA LUTEA AND IMPLANTATION SITES COMPARED USING DUNNETT'S TEST
** = Significantly different from the control group at 0.01

TABLE 1.15 SUMMARY OF FETUSES AND LITTERS WITH MALFORMATIONS [ABSOLUTE NO.]
DAY 21
	F E T U S E S				L I T T E R S
DOSE GROUP:	1	2	3	4	1	2	3	4
NUMBER EXAMINED EXTERNALLY	245	233	260	240	22	22	22	21
TAIL- ABSENT, SHORT OR FILAMENTOUS	0	1	0	0	0	1	0	0
ANUS- ATRESIA	0	1	0	0	0	1	0	0
NUMBER EXAMINED VISCERALLY	124	117	131	120	22	22	22	21
SITUS INVERSUS	0	0	1	0	0	0	1	0
HYDROCEPHALY- INTERNAL	1	0	0	0	1	0	0	0
NUMBER EXAMINED SKELETALLY	121	117	129	120	22	22	22	21
BENT LIMB BONE(S)	1	1	0	0	1	1	0	0
VERTEBRAL ANOMALY WITH OR WITHOUT ASSOCIATED RIB ANOMALY	1	0	0	0	1	0	0	0
VERTEBRAL CENTRA ANOMALY	0	1	0	0	0	1	0	0
TOTAL NUMBER WITH MALFORMATIONS
EXTERNAL :	0	1	0	0	0	1	0	0
SOFT TISSUE :	1	0	1	0	1	0	1	0
SKELETAL :	2	1	0	0	2	1	0	0
COMBINED :	3	1	1	0	3	1	1	0
1- 0 MG/KG, 2- 25 MG/KG, 3- 75 MG/KG, 4- 250 MG/KG

TABLE 1.16 SUMMARY OF LITTER PROPORTIONS OF MALFORMATIONS (% PER LITTER)
DAY 21
DOSE GROUP:		1	2	3	4
NUMBER OF LITTERS EXAMINED EXTERNALLY		22	22	22	21
TAIL- ABSENT, SHORT OR FILAMENTOUS	MEAN	0	0.6	0	0
	S.D.	0	3.05	0	0
ANUS- ATRESIA	MEAN	0	0.6	0	0
	S.D.	0	3.05	0	0
SITUS INVERSUS	MEAN	0	0	0.8	0
	S.D.	0	0	3.55	0
HYDROCEPHALY- INTERNAL	MEAN	0.9	0	0	0
	S.D.	4.26	0	0	0
NUMBER OF LITTERS EXAMINED VISCERALLY		22	22	22	21
SITUS INVERSUS	MEAN	0	0	0.8	0
	S.D.	0	0	3.55	0
HYDROCEPHALY- INTERNAL	MEAN	0.9	0	0	0
	S.D.	4.26	0	0	0
NUMBER OF LITTERS EXAMINED SKELETALLY		22	22	22	21
BENT LIMB BONE(S)	MEAN	0.9	1.1	0	0
	S.D.	4.26	5.33	0	0
VERTEBRAL ANOMALY WITH OR WITHOUT ASSOCIATED RIB ANOMALY	MEAN	1.1	0	0	0
	S.D.	5.33	0	0	0
VERTEBRAL CENTRA ANOMALY	MEAN	0	1.1	0	0
	S.D.	0	5.33	0	0
NUMBER OF LITTERS EXAMINED		22	22	22	21
TOTAL MALFORMATIONS
PERCENT PER LITTER WITH EXTERNAL MALFORMATIONS	MEAN	0	0.6	0	0
	S.D.	0	3.05	0	0
PERCENT PER LITTER WITH SOFT TISSUE MALFORMATIONS	MEAN	0.9	0	0.8	0
	S.D.	4.26	0	3.55	0
PERCENT PER LITTER WITH SKELETAL MALFORMATIONS	MEAN	2	1.1	0	0
	S.D.	6.67	5.33	0	0
TOTAL PERCENT PER LITTER WITH MALFORMATIONS	MEAN	3	0.6	0.8	0
	S.D.	7.66	3.05	3.55	0
1- 0 MG/KG, 2- 25 MG/KG, 3- 75 MG/KG, 4- 250 MG/KG
None significantly different from control group

TABLE 1.17 SUMMARY OF FETUSES AND LITTERS WITH VARIATIONS [ABSOLUTE NO.]
DAY 21
	F E T U S E S				L I T T E R S
DOSE GROUP:	1	2	3	4	1	2	3	4
NUMBER EXAMINED EXTERNALLY	245	233	260	240	22	22	22	21
NUMBER WITH FINDINGS	0	0	0	0	0	0	0	0
NUMBER EXAMINED VISCERALLY	124	117	131	120	22	22	22	21
LIVER- SMALL SUPERNUMERARY LOBE(S)	1	0	2	5	1	0	2	5
NUMBER EXAMINED SKELETALLY	121	117	129	120	22	22	22	21
14TH RUDIMENTARY RIB(S)	69	56	77	54	22	19	21	20
BENT RIB(S)	9	6	5	0	8	6	4	0
STERNUM- SUPERNUMERARY OSSIFICATION SITE	1	0	1	0	1	0	1	0
STERNEBRA(E) MALALIGNED	10	9	8	10	10	7	7	7
7TH CERVICAL OSSIFICATION SITE(S)	3	7	8	13	3	6	7	10
14TH FULL RIB(S)	13	9	11	6	8	5	5	4
PELVIC GIRDLE- CAUDAL SHIFT	2	7	19	4	2	5	8	4
STERNEBRA(E) #5 AND/OR #6 UNOSSIFIED	0	1	0	0	0	1	0	0
METACARPAL(S) AND/OR METATARSAL(S) UNOSSIFIED	1	3	6	3	1	3	3	2
REDUCED OSSIFICATION OF THE SKULL	12	7	4	0	8	4	3	0
STERNEBRA(E)- BRANCHED	1	2	2	2	1	2	2	1
VERTEBRAL CENTRA- REDUCED OSSIFICATION	2	0	0	0	1	0	0	0
1- 0 MG/KG, 2- 25 MG/KG, 3- 75 MG/KG, 4- 250 MG/KG

TABLE 1.18 SUMMARY OF LITTER PROPORTIONS OF VARIATIONS (% PER LITTER)
DAY 21
DOSE GROUP:		1	2	3	4
NUMBER OF LITTERS EXAMINED EXTERNALLY		22	22	22	21
NUMBER OF LITTERS WITH FINDINGS		0	0	0	0
NUMBER OF LITTERS EXAMINED VISCERALLY		22	22	22	21
LIVER- SMALL SUPERNUMERARY LOBE(S)	MEAN	0.8	0	1.5	3.8
	S.D.	3.55	0	4.9	6.96
NUMBER OF LITTERS EXAMINED SKELETALLY		22	22	22	21
14TH RUDIMENTARY RIB(S)	MEAN	56.2	46.5	61.4	45.3
	S.D.	20.33	29.26	23.41	21.88
BENT RIB(S)	MEAN	7.8	6.3	4.3	0.0**
	S.D.	11.61	10.84	9.37	0
STERNUM- SUPERNUMERARY OSSIFICATION SITE	MEAN	1.1	0	0.8	0
	S.D.	5.33	0	3.55	0
STERNEBRA(E) MALALIGNED	MEAN	8.4	6.8	6.7	8.1
	S.D.	9.6	10.92	11.06	14.66
7TH CERVICAL OSSIFICATION SITE(S)	MEAN	2.8	6.4	6	10
	S.D.	7.34	11.31	9.58	12.84
14TH FULL RIB(S)	MEAN	10.3	6.2	7.8	5.2
	S.D.	15.96	12.53	19.41	11.86
PELVIC GIRDLE- CAUDAL SHIFT	MEAN	1.8	4.9	13.5	3.6
	S.D.	5.88	9.72	24.18	7.72
STERNEBRA(E) #5 AND/OR #6 UNOSSIFIED	MEAN	0	1.1	0	0
	S.D.	0	5.33	0	0
METACARPAL(S) AND/OR METATARSAL(S) UNOSSIFIED	MEAN	0.9	2.3	5	2.3
	S.D.	4.26	6.03	17.35	7.43
REDUCED OSSIFICATION OF THE SKULL	MEAN	9.3	7.6	2.9	0.0**
	S.D.	14.33	17.97	7.69	0
NUMBER OF LITTERS EXAMINED SKELETALLY		22	22	22	21
STERNEBRA(E)- BRANCHED	MEAN	0.8	1.6	1.8	1.9
	S.D.	3.55	5.12	6.01	8.73
VERTEBRAL CENTRA- REDUCED OSSIFICATION	MEAN	1.3	0	0	0
	S.D.	6.09	0	0	0
NUMBER OF LITTERS EXAMINED
TOTAL VARIATIONS
PERCENT PER LITTER WITH EXTERNAL VARIATIONS	MEAN	0	0	0	0
	S.D.	0	0	0	0
PERCENT PER LITTER WITH SOFT TISSUE VARIATIONS	MEAN	0.8	0	1.5	3.8
	S.D.	3.55	0	4.9	6.96
PERCENT PER LITTER WITH SKELETAL VARIATIONS	MEAN	76.2	65.6	78.9	60.6*
	S.D.	19.87	28.95	18.87	23.31
TOTAL PERCENT PER LITTER WITH VARIATIONS	MEAN	77	65.6	80.4	64.3
	S.D.	20.56	28.95	21.14	23.56
1- 0 MG/KG, 2- 25 MG/KG, 3- 75 MG/KG, 4- 250 MG/KG
* = Significantly different from the control group at 0.05

Conclusions:

In this GLP compliant OECD 414 study, time-mated female Wistar Han rats were treated with the test substance from day 6 to 20 post-coitum. Based on the results in this prenatal developmental toxicity study the following No Observed Adverse Effect Levels (NOAEL) for the test substance were derived:
Maternal systemic NOAEL: 75 mg/kg bw/day, based on adverse changes in body weight and food consumption at 250 mg/kg bw/day.
Maternal developmental NOAEL: => 250 mg/kg bw/day
Developmental NOAEL: 75 mg/kg bw/day, based on reduced fetal body weight at 250 mg/kg bw/day.

Executive summary:

In this GLP compliant OECD 414 study, the potential of the test substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis was determined. In addition, maternal toxicity the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. Time-mated female Wistar Han rats were exposed via oral gavage from Day 6 to 20 post-coitum, inclusive. The dose levels in this study were selected to be 25, 75 and 250 mg/kg, based on the results of the dose range finder. The rats of the control group received the vehicle (4 mL/kg bw), corn oil, alone. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, organ weights, number of corpora lutea, (gravid) uterine weight and uterine contents. In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, external, visceral and skeletal malformations and developmental variations.

Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously. Body weight (gain) and food consumption was reduced in pregnant females at 250 mg/kg. In addition, body weight gain corrected for uterus weights was statistically significantly lower in these high dose females in comparison to those in the concurrent control group (2.9% vs. 14%). The combination of these differences in body weight (gain) and food consumption was considered to be adverse. Mean combined fetal body weight was decreased with 8% at 250 mg/kg when compared with concurrent controls. Retardation of growth was not observed (e.g. reduced ossification) in fetuses at 250 mg/kg, however as the mean combined fetal weight was below historical control range, this decrease was considered to be adverse. It should be noted that this effect is observed at the same dose level for which adverse findings were noted in body weights of the maternal animals. No further adverse changes were noted within this study, however there were some treatment related changes which were considered non-adverse as described below. Body weight gain and food consumption was also reduced in females at 75 mg/kg. As the decrease in mean body weight remained minimal (< 5%), recovery was noted in food consumption and the difference in body weight gain corrected for uterus weights was less marked when compared with concurrent control (6.3% vs. 14%), these differences were considered to be non-adverse. At 25 mg/kg, no toxicological differences were noted in body weight or food consumption. Piloerection was noted in 1/22, 2/22 and 4/22 females in the control, 75 and 250 mg/kg groups, respectively. In addition, 2/22 and 4/22 females at 250 mg/kg showed pale faeces and salivation, respectively. As these symptoms were transient in most females and no other symptoms of toxicity were observed, these clinical signs were regarded as non-adverse. No treatment-related changes were noted in any of the remaining parameters investigated in this study.

In conclusion, based on the results in this prenatal developmental toxicity study the following No Observed Adverse Effect Levels (NOAEL) for the test substance were derived: Maternal systemic NOAEL: 75 mg/kg, based on adverse changes in body weight and food consumption at 250 mg/kg. Maternal developmental NOAEL: => 250 mg/kg bw/day. Developmental NOAEL: 75 mg/kg, based on reduced fetal body weight at 250 mg/kg. 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

A guideline study (comparable to OECD 414) performed in compliance with GLP

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental toxicity study in rats (2018):

In this GLP compliant OECD 414 study, the potential of the test substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis was determined. In addition, maternal toxicity the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. Time-mated female Wistar Han rats were exposed via oral gavage from Day 6 to 20 post-coitum, inclusive.The dose levels in this study were selected to be 25, 75 and 250 mg/kg, based on the results of the dose range finder. The rats of the control group received the vehicle (4 mL/kg bw), corn oil, alone. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, organ weights, number of corpora lutea, (gravid) uterine weight and uterine contents. In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, external, visceral and skeletal malformations and developmental variations.

Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously. Body weight (gain) and food consumption was reduced in pregnant females at 250 mg/kg. In addition, body weight gain corrected for uterus weights was statistically significantly lower in these high dose females in comparison to those in the concurrent control group (2.9% vs. 14%). The combination of these differences in body weight (gain) and food consumption was considered to be adverse. Mean combined fetal body weight was decreased with 8% at 250 mg/kg when compared with concurrent controls. Retardation of growth was not observed (e.g. reduced ossification) in fetuses at 250 mg/kg, however as the mean combined fetal weight was below historical control range, this decrease was considered to be adverse. It should be noted that this effect is observed at the same dose level for which adverse findings were noted in body weights of the maternal animals. No further adverse changes were noted within this study, however there were some treatment related changes which were considered non-adverse as described below. Body weight gain and food consumption was also reduced in females at 75 mg/kg. As the decrease in mean body weight remained minimal (< 5%), recovery was noted in food consumption and the difference in body weight gain corrected for uterus weights was less marked when compared with concurrent control (6.3% vs. 14%), these differences were considered to be non-adverse. At 25 mg/kg, no toxicological differences were noted in body weight or food consumption. Piloerection was noted in 1/22, 2/22 and 4/22 females in the control, 75 and 250 mg/kg groups, respectively. In addition, 2/22 and 4/22 females at 250 mg/kg showed pale faeces and salivation, respectively. As these symptoms were transient in most females and no other symptoms of toxicity were observed, these clinical signs were regarded as non-adverse. No treatment-related changes were noted in any of the remaining parameters investigated in this study.

In conclusion, based on the results in this prenatal developmental toxicity study the following No Observed Adverse Effect Levels (NOAEL) for the test substance were derived: Maternal systemic NOAEL: 75 mg/kg, based on adverse changes in body weight and food consumption at 250 mg/kg. Maternal developmental NOAEL: => 250 mg/kg bw/day. Developmental NOAEL: 75 mg/kg, based on reduced fetal body weight at 250 mg/kg. 

Developmental toxicity study in rabbits (2021):

The objectives of this study were to determine the potential of the test substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given via diet to time-mated female New Zealand White rabbits from Days 7 to 29 post-coitum, inclusive according to OECD TG 414 and GLP principles. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels in this study were selected to be 0, 2220, 3330, 5000 ppm (corresponding to a mean actual test item intake of 65, 101 and 125 mg/kg/day, respectively), based on the results of the Dose Range Finder. Chemical analyses of dietary preparations were conducted once during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, test item intake, macroscopic examination, organ weights, uterine contents, corpora lutea, implantation sites, and pre- and post-implantation loss. In addition, the following parameters were determined for the F1-generation: the number of live and dead foetuses, foetal body weights, sex ratio, external, visceral and skeletal malformations and developmental variations.

Results showed for maternal toxicity that no mortality occurred during the study period. At 5000 ppm (corresponding to an actual test item intake of 125 mg/kg/day), a lower mean food consumption (absolute and relative) was observed from post-coitum Day 7 onwards. This was accompanied with a reduced body weight gain during the entire treatment period and an increased incidence and severity of reduced faeces production. Additionally, body weight loss corrected for gravid uterus was lower compared to control (-250.4vs-104.8 gram in controls; 4.2% lower corrected weight gain in percent of weight on post-coitum Day 7 compared to control). As a dose-dependent reduced body weight gain and decreased food consumption were also noted in the Dose Range Finder at actual test item intake levels of 151 mg/kg/day (6700 ppm; intended dose level of 300 mg/kg/day) and 161 mg/kg/day (10050 ppm; intended dose level of 600 mg/kg/day), together with early deliveries of 3/6 females at 151 and 4/6 females at 161 mg/kg/day, these effects were considered to have a very steep dose-response relation. For this reason, although minor effects on body weight and food consumption were noted in the Main study, these effects cannot be neglected and were considered to be adverse. No test item-related gross findings were observed at necropsy. The number of corpora lutea, implantation sites, viable or dead foetuses, early or late resorptions, pre- and post-implantation loss were considered not affected by treatment with the test item up to 5000 ppm. For developmental toxicity, mean male, female and combined (male and female) foetal weights were lower in all treatment groups at 2220, 3330 and 5000 ppm when compared with controls. As mean foetal weights (per litter) at 5000 ppm were below the range of the historical control data, this decrease was considered to be adverse at this concentration. In addition, higher litter incidences of the skeletal variations unossified tarsals and unossified pubis were noted at 5000 ppm. Although statistical significance was only reached for the increased incidence of unossified pubis at 5000 ppm, the higher litter incidences of both unossified tarsals and unossified pubis at this concentration were outside the range of historical control data. Based on the individual lower foetal weights of most affected foetuses, this indication of a delayed ossification in most foetuses was considered a foetal weight effect and not a direct toxicological effect of the test substance. A dose related increase in mean litter incidence of ovary cysts was observed in all dose groups, with values above the historical control range at 5000 ppm, although not statistically significant. However, as this concerns a variation with no effect on development, it was considered to be non-adverse. No test item-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, malformations and external developmental variations).

In conclusion, the maternal NOAEL is set at 3330 ppm (corresponding to an actual test item intake of 101 mg/kg/day), based on the decreased body weight gain and food consumption. The developmental NOAEL is set at 3330 ppm (corresponding to an actual test substance intake of 101 mg/kg/day), based on the decreased mean foetal weights at 5000 ppm. A higher dose level than 5000 ppm was considered not feasible as a dose level of 6700 ppm resulted in a high incidence of early deliveries during the Dose Range Finder study

Justification for classification or non-classification

Based on the available information, classification is not required for the test substance in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.