Zinc bis(dibutyldithiocarbamate)

Developmental toxicity / teratogenicity

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

15 Oct 2020 to 13 Nov 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

A Dose Range Finder (Test Facility Reference No. 20247938) was conducted to select dose levels for the Main study (Test Facility Study No. 20247940). No guidelines are applicable as this study was used for dose level selection purposes only. If not mentioned otherwise, test system, procedures and techniques were identical to those used during the Main study.

GLP compliance:

no

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: 17-19 weeks old
- Weight at study initiation: 2898 and 4145 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
- 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: 19 to 20 °C
- Humidity (%): Targeted: 40 to 70, actual mean: 47 to 75

IN-LIFE DATES: 15 Oct 2020 to 13 Nov 2020

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 (frequency): Diets were prepared at least once weekly
- Mixing appropriate amounts with (Type of food):
- 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:

no

Details on analytical verification of doses or concentrations:

Samples for diet analysis were not collected during the DRF, as concentration, homogeneity, and stability analysis was not performed.
However, to limit the impact, the diet preparation was performed with approved procedures and documented in detail.
Homogeneity and accuracy of the diet under test conditions was demonstrated in the analytical method development and validation study.

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.:

3 350 ppm

Remarks:

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

Dose / conc.:

6 700 ppm

Remarks:

Corresponded with an intended dose level of approximately 300 mg/kg/day

Dose / conc.:

10 050 ppm

Remarks:

Corresponded with an intended dose level of approximately 600 mg/kg/day

No. of animals per sex per dose:

6 females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a non-GLP tolerability study with the test substance in rabbits by dietary administration.

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:

All live fetuses were euthanized by administration of sodium pentobarbital into the oral cavity using a small metal feeding tube.
Each viable fetus of animals surviving to planned necropsy was externally examined in detail and weighed. For late resorptions, a gross external examination was performed.
Fetuses of animals sacrificed before scheduled necropsy were externally examined in detail and euthanized by sodium pentobarbital.
No visceral (internal) or skeletal examination was performed.
A malformed fetus (No. A015-5 (control)) was collected and fixed in the most appropriate fixative (based on type of malformation). Other fetuses and late resorptions without malformations were discarded.

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 analysed 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 foetuses, 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 foetuses, early and late resorptions, and pre- and post-implantation 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
Post- implantation 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 foetuses 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 foetuses as a mean litter proportion on a total group basis, where:
Viable foetuses affected/litter (%): number of viable foetuses affected/litter / number of viable foetuses/litter x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Reduced faeces production was observed in all females of the control, 3350, 6700 and 10050 ppm groups, 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 10050 ppm

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

In total, nine females were sacrificed prior to scheduled necropsy, of which seven cases of early delivery are most probably related to treatment with the test item. Details can be found in the 'Any other information results incl. tables' field.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 6700 and 10050 ppm, body weight gain and mean food consumption were decreased during the entire treatment period (not always statistically significant). In all treated groups, mean weight gains corrected for gravid uterus were lower compared to control, however without reaching statistical significance and without a clear dose-response.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Achieved mean over means intake were 89, 151 and 161 mg/kg bw/day for the 3350, 6700 and 10050 ppm dose group levels.

Food efficiency:

no effects observed

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

not examined

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 necropsy did not reveal any alterations that were considered to be toxicologically relevant.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

In general, early deliveries may be observed when rabbits eat less than 15 gram/day during the final trimester of gestation (Matsuoka et. al.) (such as Female No. 3). In this study, all animals with an early delivery treated at 6700 and 10050 ppm showed a lower average food consumption during the entire post-coitum period when compared to controls. This may indicate a relationship between the overall low food consumption during pregnancy and early deliveries. However, as some animals with a low average of total food consumption in these groups did not deliver early and no historical control data are available for this, these cases of early delivery were considered most probably related to treatment with the test-item. In the 0 ppm group, 1/6 female showed early delivery (16.7%) and in the 3350 ppm group, 0/6 female showed early delivery (0%). In the 6700 ppm group, 3/6 females showed early delivery (50%) and in the 10050 ppm group 4/6 females (66.7%).

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The number of pre- and post-implantation loss in all treated groups were similar to the control group and in the range of normal biological variation.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

The number of early and late resorptions in all treated groups were similar to the control group and in the range of normal biological variation.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All females, including those that were necropsied preterm, were found to be pregnant, except for Female Nos. 5 (control) and 10 (3350 ppm). Excluding the females that were sacrificed prior to scheduled necropsy, delivered early and the non-pregnant females resulted in a total of three, five, three and two pregnant females for evaluation in the control, 3350, 6700 and 10050 ppm groups, respectively. As the remaining number of pregnant females was low in this study, only limited evaluation of developmental data could be performed. However, evaluation was sufficient to select dose levels for the Main study, which was the objective of this DRF.

Other effects:

no effects observed

Description (incidence and severity):

The number of corpora lutea in all treated groups were similar to the control group and in the range of normal biological variation.
Mean numbers of implantation sites were lower at 10050 ppm compared to controls (8.0 vs 9.3 in controls; below the range of historical control data).

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Foetal body weight was decreased in all treatment group as compared to control. At 10050 ppm, mean foetal weight was below the range of historical control data, whereas foetal body weight in controls was relatively high. At 3350 ppm and 6700 ppm, foetal body weight remained within the historical control range.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Litter sizes were 8.7, 10.2, 11.3 and 7.5 fetuses/litter for the control and 3350, 6700 and 10050 ppm groups, respectively

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

External examination of the foetuses did not show any treatment-related abnormalities.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Maternal toxicity findings

- Female Nos. 16, 17 and 18 (6700 ppm) were sacrificed as they started to deliver their offspring on post-coitum Days 28 or 29. All females had a low food consumption during the entire treatment period. Female No. 16 was observed with reduced faeces production, lean appearance and scabs and necropsy revealed an enlarged gallbladder, oviducts with cysts and an emaciated appearance. Female Nos. 17 and 18 were observed with reduced faeces production and necropsy revealed no findings.

- Female Nos. 20, 21, 22 and 24 (10050 ppm) were sacrificed as they started to deliver their offspring on post-coitum Days 25 or 26. All females had a low food consumption during the entire treatment period together with a reduced faeces production. At necropsy, Female No. 20 was noted with an enlarged gallbladder and pelvic dilation of the kidneys and Female No. 24 showed an enlarged gallbladder, an emaciated appearance and a watery-cloudy, reddish content in the urinary bladder. Macroscopic examination revealed no findings for Female Nos. 21 and 22.

 

The following early sacrifices occurred in the control group and were not related to treatment with the test item:

- Female No. 2 (control) was sacrificed in extremis on post-coitum Day 27 based on the absence of food consumption for 7 consecutive days together with body weight loss (5%) compared to post-coitum Day 7 and clinical observations (i.e. reduced faeces production (grade 3), lean appearance, red fluid on manure tray). At necropsy, a thickened gallbladder wall with greenish foci was noted and this female was emaciated.

- Female No. 3 (control) was sacrificed on post-coitum Day 29 as it started to deliver its offspring prior to necropsy. This female was noted with absent food consumption from post-coitum Day 21 onwards together with body weight loss (9%) on post-coitum Day 29 prior to its early delivery. Except for reduced faeces production, no clinical observations were noted. Macroscopic examination revealed no abnormalities.

Table 1. Body weights (gram) summary F) generation females

		Group 1 Control	Group 2 3350 ppm	Group 3 6700 ppm	Group 4 10050 ppm
DAY 0	MEAN	3511	3676	3490	3307
	ST.DEV.	369.7	437.5	345.8	77.3
	N	5	5	6	6
DAY 7	MEAN	3461	3576	3388	3177
	ST.DEV.	328.4	358.5	276.4	172.1
	N	5	5	6	6
DAY 9	MEAN	3476	3605	3369	3154
	ST.DEV.	337.0	379.7	248.7	149.6
	N	5	5	6	6
DAY 12	MEAN	3543	3673	3391	3175
	ST.DEV.	325.1	366.9	249.9	121.0
	N	5	5	6	6
DAY 15	MEAN	3625	3724	3437	3205
	ST.DEV.	323.7	358.1	221.1	180.7
	N	5	5	6	6
DAY 18	MEAN	3705	3811	3459	3212 *
	ST.DEV.	280.2	389.0	217.5	180.0
	N	5	5	6	6
DAY 21	MEAN	3750	3847	3481	3238 *
	ST.DEV.	281.6	411.3	215.0	146.3
	N	5	5	6	6
DAY 24	MEAN	3722	3837	3504	3255 *
	ST.DEV.	255.5	432.7	192.0	131.0
	N	5	5	6	6
DAY 27	MEAN	3689	3907	3503	3343
	ST.DEV.	243.8	423.4	217.3	137.9
	N	5	5	6	2
DAY 29	MEAN	3600	3917	3546	3318
	ST.DEV.	380.7	442.3	267.0	169.7
	N	4	5	4	2

*/** 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 3350 ppm	Group 3 6700 ppm	Group 4 10050 ppm
POSTCOITUM DAY 7	MEAN	0	0	0	0
	ST.DEV.	0.0	0.0	0.0	0.0
	N	5	5	6	6
DAY 9	MEAN	0	1	0	-1
	ST.DEV.	0.8	0.8	0.9	1.1
	N	5	5	6	6
DAY 12	MEAN	2	3	0	0
	ST.DEV.	0.7	1.0	1.3	3.1
	N	5	5	6	6
DAY 15	MEAN	5	4	2	1 *
	ST.DEV.	1.3	1.5	2.9	2.9
	N	5	5	6	6
DAY 18	MEAN	7	7	2	1 *
	ST.DEV.	3.4	1.9	3.9	4.3
	N	5	5	6	6
DAY 21	MEAN	9	8	3	2 *
	ST.DEV.	3.9	3.1	4.2	3.5
	N	5	5	6	6
DAY 24	MEAN	8	7	4	3
	ST.DEV.	4.8	3.4	3.9	4.7
	N	5	5	6	6
DAY 27	MEAN	7	9	4	0
	ST.DEV.	8.2	3.9	3.8	2.3
	N	5	5	6	2
DAY 29	MEAN	7	10	3	-1
	ST.DEV.	11.4	5.5	6.2	3.3
	N	4	5	4	2

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 3. Summary of foetal data at scheduled necropsy

Group		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	26	0	2	0	2	28	32	4	NA	3
	MEAN	8.7	0.0	0.7	0.0	0.7	9.3	10.7	1.3	44.3
	S.D.	0.58	0.00	1.15	0.00	1.15	1.53	1.53	0.58	0.50
2	TOTAL	51	0	1	0	1	52	54	2	NA	5
	MEAN	10.2	0.0	0.2	0.0	0.2	10.4	10.8	0.4	39.6
	S.D.	1.64	0.00	0.45	0.00	0.45	1.67	1.92	0.55	2.25
3	TOTAL	34	0	1	1	2	36	37	1	NA	3
	MEAN	11.3	0.0	0.3	0.3	0.7	12.0	12.3	0.3	37.6
	S.D.	2.08	0.00	0.58	0.58	1.15	2.65	2.08	0.58	7.13
4	TOTAL	15	0	1	0	1	16	22	6	NA	2
	MEAN	7.5	0.0	0.5	0.0	0.5	8.0	11.0	3.0	33.5
	S.D.	3.54	0.00	0.71	0.00	0.71	4.24	0.00	4.24	4.67

Conclusions:

Due to the high incidence of females that delivered early at 6700 and 10050 ppm (3/6 and 4/6 females, respectively) and the high risk of ending up with an insufficient number of animals in the Main study for evaluation of developmental parameters, a dose level of 5000 ppm was selected as top dose level for the Main study. Based on the results of the Dose Range Finder, selected dose levels for the Main study were 2220, 3330 and 5000 ppm.

Executive summary:

The objectives of this study were to determine the dose levels for the main study investigating 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. No guidelines are applicable as this study was used for dose level selection purposes only. The following dose levels were selected: 0, 3350, 6700 and 10050 ppm corresponded with an intended dose level of approximately, 100, 300 and 600 mg/kg/day, respectively.

Results showed in total, nine females were sacrificed prior to scheduled necropsy, of which seven cases of early delivery are most probably related to treatment with the test item. In general, early deliveries may be observed when rabbits eat less than 15 gram/day during the final trimester of gestation (Matsuoka et. Al). In this study, all animals with an early delivery treated at 6700 and 10050 ppm showed a lower average food consumption during the entire post-coitum period when compared to controls. This may indicate a relationship between the overall low food consumption during pregnancy and early deliveries. However, as some animals with a low average of total food consumption in these groups did not deliver early and no historical control data are available for this, these cases of early delivery were considered most probably related to treatment with the test-item. Macroscopic observations at necropsy did not reveal any alterations that were considered to be toxicologically relevant. The number of corpora lutea, pre- and post-implantation loss, early and late resorptions in all treated groups were similar to the control group and in the range of normal biological variation. Foetal body weight was decreased in all treatment group as compared to control. At 10050 ppm, mean foetal weight was below the range of historical control data, whereas foetal body weight in controls was relatively high. At 3350 ppm and 6700 ppm, foetal body weight remained within the historical control range. External examination of the foetuses did not show any treatment-related abnormalities. Due to the high incidence of females that delivered early at 6700 and 10050 ppm (3/6 and 4/6 females, respectively) and the high risk of ending up with an insufficient number of animals in the Main study for evaluation of developmental parameters, a dose level of 5000 ppm was selected as top dose level for the Main study.

In conclusion, based on the results of the Dose Range Finder, selected dose levels for the Main study were 2220, 3330 and 5000 ppm.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

Zinc bis(dibutyldithiocarbamate)

EC Number:

205-232-8

EC Name:

Zinc bis(dibutyldithiocarbamate)

Cas Number:

136-23-2

Molecular formula:

C18H36N2S4Zn

IUPAC Name:

zinc bis(dibutyldithiocarbamate)

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 220 ppm

Remarks:

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

Doses / concentrations 2

Dose / conc.:

3 330 ppm

Remarks:

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

Doses / concentrations 3

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

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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

Effect levels (maternal animals)

1

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

2

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.

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

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.

Effect levels (fetuses)

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

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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