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Toxicological information

Developmental toxicity / teratogenicity

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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese MAFF 59 NohSan No. 4200, "Teratogenicity Study" January 1985
Deviations:
no
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

Constituent 1
Chemical structure
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)

open allclose all
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
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.

 

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