1-(allyloxy)-2-methyl-1-oxopropan-2-yl 2-chloro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]benzoate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 August 1995 to 19 February 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed to GLP and in line with the standardised guidelines OECD 414, EC OJ No L133/24, Japanese MAFF 59 and US EPA OPP 83 -3, with no deviations thought to impact the reliability of the presented results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF 59 NohSan No. 4200, "Teratogenicity Study" January 1985

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: European Communities Commission Directive 87/302/EEC, "Teratogenicity Study - Rodent and Non-Rodent", OJ No L133/24, May 30, 1988

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1-(allyloxy)-2-methyl-1-oxopropan-2-yl 2-chloro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]benzoate

EC Number:

603-837-5

Cas Number:

134605-64-4

Molecular formula:

C20H18ClF3N2O6

IUPAC Name:

1-(allyloxy)-2-methyl-1-oxopropan-2-yl 2-chloro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]benzoate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Physical state: Solid, tan coloured powder
- Storage condition of test material: Room temperature

Test animals

Species:

rabbit

Strain:

other: Russian Chbb:HM

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 3 months (minimum)
- Fasting period before study: Not reported
- Housing: housed individually in battery cages with steel slatted floors.
- Diet (e.g. ad libitum): Pelleted, certified standard feed, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 ± 2 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): about 16 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: From: 11/09/95 To: 29/02/96

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% carboxymethylcellulose and 0.1 % aqueous polysorbate 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test substance-vehicle mixtures were prepared daily with a high-speed homogeniser. Homogeneity of the mixtures during administration was maintained with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 2.5, 25 and 250 mg/mL mixture
- Amount of vehicle (if gavage): 4 mL/kg of actual bodyweight
- Purity: 0.5% (w/w) aqueous solution of sodium carboxymethylcellulose and 0.1% (w/w) aqueous polysorbate 80

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In order to permit determination of content, homogeneity and stability of the test substance under the actual conditions of administration during the study, samples of test substance-vehicle mixtures were taken on the date(s) designated below, once before and once after dosing. The samples from before dosing were taken from the top, middle and bottom of the container; the samples from after dosing were taken from the middle of the container.
Samples were taken in duplicate. Together with 10 mL of vehicle and approximately 2.0 g of test substance they were frozen until analysis.
- dates of sampling: 21 and 27 September 1995

Samples were analysed by HPLC with the following conditions:
- Column: 5 µm; 125 mm x 4.6 mm (o.d.)
- Temperature: room temperature
- Eluent: Acetonitrile/0.02 M H3PO4 (60+40 v/v)
- Flow: 1.0 mL/min
- Wavelength: 274 nm
- Injection volume: 10 μL

Details on mating procedure:

- Impregnation procedure: Females were artificially inseminated with diluted semen from bucks of the same strain. The day of insemination was designated as day 0 of pregnancy.

Duration of treatment / exposure:

From day 7 to day 19 of gestation.

Frequency of treatment:

Daily

Duration of test:

29 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 10, 100 and 1000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

20 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a previous rangefinding study in pregnant rabbits (study no. 951036). In the preliminary study, test substance was non-toxic to dams, embryos and foetuses at doses up to 1000 mg/kg, and there was no indication of teratogenesis.
- Rationale for animal assignment (if not random): Inseminated females were allocated to experimental and control groups using a method of randomisation by weight stratification.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (> 6 hours apart)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION: Yes (on days 4, 7, 12, 16, 20, 24 and 29)
- Food consumption for each animal determined according to the following formula: feed comsumption (g) per period / days per period

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: Macroscopic pathological examination of the main organs of the thoracic and abdominal cavities, in particular the genitals.

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 of litter
- Visceral examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

Statistical analysis of continuous data (e.g. body weight, feed consumption) was performed using the Analysis of Variance Procedure (ANOVA) followed by Dunnett's t-Test in case of a significant result in the ANOVA. Categorical data (e.g. malformation counts) were analysed using Chi-Square test followed by Fisher's Exact test in case of a significant result in the Chi-Square test.
Non-parametric data (e.g. mean percent affected fetuses/litter) were analysed using the Kruskal-Wallis nonparametric analysis of variance test followed by Mann-Whitney U-test.

Historical control data:

Available and documented within the study report.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
- Maternal Toxicity
None of the maternal animals died during the study and only occasional incidental clinical signs occurred during the study.
At 1000 mg/kg, there was a slight reduction in food consumption during the dosing period (days 7 to 20) and an increase in food consumption at the end of gestation when the animals were no longer dosed (days 20 to 29). In this group a significant difference in weight change was seen only for days 12 to 16 of gestation and overall on days 7 to 19. Overall weight change for days 7 to 29 was comparable among all groups.
Gravid uterus weights, carcass weights, and net weight change from day 7 were similar in all four groups. There were no maternal necropsy findings.

- Reproduction and Cesarean Section Data
Preimplantation losses, numbers of implantation sites and live litter size were similar in all groups.
At 1000 mg/kg, there was an increase in the incidence of post implantation loss, almost exclusively in the form of early resorptions. Three females in this group showed total early embryonic resorption; for the remaining 16 pregnant females, this parameter was increased when compared to controls and historical control data.

Effect levels (maternal animals)

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
At 1000 mg/kg, foetal weights (both male and female) were slightly reduced compared to controls.
The incidence and type of external, visceral and skeletal findings were not affected by treatment.

Effect levels (fetuses)

1

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

2

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Analytical results:

The chemical analysis determined the concentration, homogeneity and stability of the test material in 0.1% (w/w) aqueous polysorbate 80 with 0.5% (w/w) CMC by HPLC-analysis. The overall mean concentration of the homogeneity samples were found to be 92.7%, 92.6%, and 99.0% of the nominal concentrations for dose group 2 (2.5 mg/mL), for dose group 3 (25 mg/mL), and for dose group 4 (250 mg/mL), respectively. The homogeneity varied in the range from -2% to +2% of the mean concentrations. The test material was found to be stable in 0.1% (w/w) aqueous polysorbate 80 with 0.5% (w/w) CMC under actual conditions of administration over the period of dosing.

Table 1: Mean Maternal Bodyweight (g)

Day	Mean Maternal Bodyweight (g)
	Group 1 (0 mg/kg bw/day)	Group 2 (10 mg/kg bw/day)	Group 3 (100 mg/kg bw/day)	Group 4 (1000 mg/kg bw/day)
0	2882	2874	2878	2892
1	2896	2890	2875	2903
2	2888	2886	2878	2902
3	2876	2887	2870	2887
4	2878	2869	2864	2882
5	2865	2865	2860	2870
6	2858	2857	2854	2865
7	2860	2855	2854	2860
8	2851	2856	2845	2863
9	2843	2851	2850	2849
10	2851	2854	2842	2842
11	2846	2844	2841	2840
12	2837	2853	2844	2828
13	2836	2864	2847	2819
14	2842	2866	2849	2829
15	2856	2873	2861	2821
16	2860	2884	2870	2818
17	2866	2888	2868	2803
18	2857	2887	2869	2790
19	2853	2879	2863	2782
20	2842	2869	2847	2763
21	2836	2866	2848	2755
22	2833	2861	2840	2763
23	2839	2864	2843	2781
24	2851	2879	2851	2805
25	2870	2882	2868	2828
26	2884	2898	2876	2843
27	2902	2906	2889	2871
28	2917	2924	2901	2902
29	2932	2943	2907	2921

 

Table 2: Mean Maternal Bodyweight Gain (g)

Days	Mean Maternal Bodyweight Change (g)
	Group 1 (0 mg/kg bw/day)	Group 2 (10 mg/kg bw/day)	Group 3 (100 mg/kg bw/day)	Group 4 (1000 mg/kg bw/day)
0-4	-4	-5	-14	-10
4-7	-19	-13	-10	-23
7-12	-23	-2	-9	-31
12-16	24	31	26	-11*
16-20	-18	-14	-23	-54
20-24	9	9	4	42
24-29	81	64	56	116
7-19	-7	23	9	-78*
7-29	72	88	53	61

 * p ≤ 0.05

Table 3: Mean Food Consumption

Days	Mean Food Consumption g/animal/day
	Group 1 (0 mg/kg bw/day)	Group 2 (10 mg/kg bw/day)	Group 3 (100 mg/kg bw/day)	Group 4 (1000 mg/kg bw/day)
0-4	91.9	95.4	87.4	91.8
4-7	82.2	89.3	87.2	87.5
7-12	83.9	86.4	86.9	71.7
12-16	64.6	73.2	69.6	49.2
16-20	63.9	71.8	70.3	42.1
20-24	69.6	76.5	71.2	81.5
24-29	80.1	75.0	79.7	105.6*

 * p≤ 0.05

Table 4: Summary of Caesarean Section Data for Pregnant Dams

Observation		Group 1 (0 mg/kg bw/day)	Group 2 (10 mg/kg bw/day)	Group 3 (100 mg/kg bw/day)	Group 4 (1000 mg/kg bw/day)
Number of pregnant animals used for calculation		19	17	18	19
Corpora lutea	Total	121	120	122	132
	Mean	6.4	7.1	6.8	6.9
Implantation Sites	Total	97	93	89	112
	Mean	5.1	5.5	4.9	5.9
Preimplantation loss % per animal	Total	24	27	33	20
	Mean %	21.3	23.1	27.5	17.2
Foetuses (per animal)	Total	83	71	77	81
	Mean	4.4	4.2	4.3	4.3
	% Alive	100	100	100	100
	% Dead	0	0	0	0
Live foetuses (per animal)	Total	83	71	77	81
	Mean	4.4	4.2	4.3	4.3
Malformed live foetuses (per animal)	Total	0	0	0	1
	Mean	0	0	0	0.1
Dead foetuses	Total	0	0	0	0
Early resorptions (per animal)	Total	14	21	12	29
	Mean	0.7	1.2	0.7	1.5
	% of implantations per animal	16.5	21.3	12.7	31.9
Late resorptions (per animal)	Total	0	1	0	2
	Mean	0	0.1	0	0.1
	% of implantations per animal	0	1.5	0	1.4
Abortion Sites	Total	0	0	0	0
Postimplantation loss (per animal)	Total	14	22	12	31
	Mean	0.7	1.3	0.7	1.6
	% of implantations per animal	16.5	22.8	12.7	33.3
Affected implants (per animal)	Total	14	22	12	32
	Mean	0.7	1.3	0.7	1.7
	% of implantations per animal	16.5	22.8	12.7	34.0
Number of litters used for calculations		18	15	18	16
Viable male foetuses	Number	39	34	40	36
	%	47.0	47.9	51.9	44.4
Viable female foetuses	Number	44	37	37	45
	%	53.0	52.1	48.1	55.6
Foetal bodyweight (g)	Mean	41.4	41.0	41.9	40.0
Male foetal bodyweight (g)	Mean	41.3	41.5	42.4	39.8
Female foetal bodyweight (g)	Mean	41.5	39.6	41.4	40.2

 

Table 5: Summary of Caesarean Section Data Day 29 Post-Coitum

Observation			Group 1 (0 mg/kg bw/day)	Group 2 (10 mg/kg bw/day)	Group 3 (100 mg/kg bw/day)	Group 4 (1000 mg/kg bw/day)
Number of pregnant animals used for calculation			18	15	18	16
Corpora lutea	Total		116	106	122	119
	Mean		6.4	7.1	6.8	7.4
Implantation Sites	Total		93	80	89	103
	Mean		5.2	5.3	4.9	6.4
Preimplantation loss % per animal	Total		23	26	33	16
	Mean %		21.4	25.2	27.5	13.7
Foetuses (per animal)	Total		83	71	77	81
	Mean		4.6	4.7	4.3	5.1
	% Alive		100	100	100	100
	% Dead		0	0	0	0
Live foetuses (per animal)	Total		83	71	77	81
	Mean		4.6	4.7	4.3	5.1
Malformed live foetuses (per animal)	Total		0	0	0	1
	Mean		0	0	0	0.1
Dead foetuses	Total		0	0	0	0
Early resorptions (per animal)	Total		10	8	12	20
	Mean		0.6	0.5	0.7	1.3
	% of implantations per animal		11.9	10.8	12.7	19.2
Late resorptions (per animal)	Total		0	1	0	2
	Mean		0.0	0.1	0.0	0.1
	% of implantations per animal		0.0	1.7	0.0	1.7
Abortion Sites	Total		0	0	0	0
Postimplantation loss (per animal)	Mean		10	9	12	22
	Total		0.6	0.6	0.7	1.4
	% of implantations per animal		11.9	12.5	12.7	20.8
Affected implants (per animal)	Total		10	9	12	23
	Mean		0.6	0.6	0.7	1.4
	% of implantations per animal		11.9	12.5	12.7	21.6
Number of litters used for calculations			18	15	18	16
Viable male foetuses		Number	39	34	40	36
		%	47.0	47.9	51.9	44.4
Viable female foetuses		Number	44	37	37	45
		%	53.0	52.1	48.1	55.6
Foetal bodyweight (g)		Mean	41.4	41.0	41.9	40.0
Male foetal bodyweight (g)		Mean	41.3	41.5	42.4	39.8
Female foetal bodyweight (g)		Mean	41.5	39.6	41.4	40.2

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, maternal toxicity (reduced food consumption and body weight gain) and embryo and fetal toxicity (increased incidence of early resorption and decreased fetal weights) were seen in the 1000 mg/kg group. The No Observed Effect Level (NOEL) of the test material in this study was therefore considered to be 100 mg/kg for dams and fetuses. There was no indication of teratogenic potential.

Executive summary:

The study was performed to meet the requirements of OECD 414, EC OJ No L133/24, Japanese MAFF 59 and US EPA OPP 83 -3 under GLP conditions. The study was performed to evaluate the potential of the test substance to cause embryotoxic, foetotoxic, and teratogenic effects in rabbits. The test substance was evaluated over a range of concentrations - 0, 10, 100 and 1000 mg/kg. Females were inseminated with semen from males of the same stock. Inseminated females were treated daily with the test material from day 7 to day 19 of gestation. On day 29 dams were sacrificed and foetuses extracted and examined for skeletal and visceral effects.

None of the maternal animals died during the study and only occasional incidental clinical signs occurred during the study.

At 1000 mg/kg, there was a slight reduction in food consumption during the dosing period (days 7 to 20) and an increase in food consumption at the end of gestation when the animals were no longer dosed (days 20 to 29). In this group a significant difference in weight change was seen only for days 12 to 16 of gestation and overall on days 7 to 19. Overall weight change for days 7 to 29 was comparable among all groups.

Gravid uterus weights, carcass weights, and net weight change from day 7 were similar in all four groups. There were no maternal necropsy findings.

Preimplantation losses, numbers of implantation sites and live litter size were similar in all groups.

At 1000 mg/kg, there was an increase in the incidence of post implantation loss, almost exclusively in the form of early resorptions. Three females in this group showed total early embryonic resorption; for the remaining 16 pregnant females, this parameter was increased when compared to controls and historical control data.

At 1000 mg/kg, foetal weights (both male and female) were slightly reduced compared to controls. However, the incidence and type of external, visceral and skeletal findings were not affected by treatment.

Under the conditions of the study, maternal toxicity (reduced food consumption and body weight gain) and embryo and foetal toxicity (increased incidence of early resorption and decreased foetal weights) were seen in the 1000 mg/kg group. The No Observed Effect Level (NOEL) of the test material in this study was therefore considered to be 100 mg/kg for dams and foetuses. There was no indication of teratogenic potential.