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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:
09 September - 31 October 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 22 January 2001
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

1
Chemical structure
Reference substance name:
6-amino-5-chloro-2-cyclopropylpyrimidine-4-carboxylic acid
EC Number:
617-769-9
Cas Number:
858956-08-8
Molecular formula:
C8H8ClN3O2
IUPAC Name:
6-amino-5-chloro-2-cyclopropylpyrimidine-4-carboxylic acid

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA
- Age at study initiation: approximately 67 days
- Weight at study initiation: 222 - 270 g
- Housing: individually in stainless steel, wire-mesh cages suspended above cage boards
- Diet: PMI Nutrition International, LLC Certified Rodent Lab Diet 5002 (pellets), ad libitum
- Water: tap water (United water Delaware), ad libitum
- Acclimation period: approximately 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 -70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The formulations of the test substance in the vehicle were prepared daily. To achieve the respective test concentrations of active ingredient, the formulations were adjusted for sample purity (92.2%).

VEHICLE
- Other: Volume administered: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation were taken 2 times near the beginning and the end of the study. Analyses addressed uniformity of mixing, concentration and stability. One vehicle sample and 3 samples (top,middle,and bottom samplings) of each concentrations were analysed to verify concentration and uniformity of mixing near study beginning and one vehicle sample and one sample of each concentration was analysed to verify concentration near end of the study. A fourth sample of each concentration was held for 5 h at room temperature and was then analysed for stability of the test substance in the formulation. Sample analyses were perfomed by Critical Path Services (CPS) and were shipped frozen. Data from the analysis of the formulation samples, including the 5-h stability sample, indicated that the test substance was uniformly mixed in the vehicle at the targeted concentrations and was stable in the vehicle under the conditions of the study. Test substance was not found in the control sample.

The COA included in the original finalised report listed a purity of 92.2% which did not fully account for all the inorganic impurities associated with this sample. Follow-up analysis of DPX-MAT28-009 was conducted after the final report had issued. The revised COA, included in this revised report, presents the purity as 90.5% and now accounts for the full impurity profile as determined in GLP analytical study. This reduction in determined purity of 1.7% had minimal impact on the reported doses determined by chemical analyses and on the values utilized for risk assessments. Therefore, the reported doses or dietary intake values have not been adjusted.

Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
Gestation Days 6 -20
Frequency of treatment:
daily
Duration of test:
21 Days
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a pilot developmental toxicity study in rats with a similar compound (methyl ester of the test substance), the test substance was administered to time-mated female Crl:CD(SD) rats (8/group) once daily on GD 6 - 20 at dosages of 25, 300, or 1000 mg/kg/day. During the in-life portion of the study, maternal body weights, food consumption, and clinical signs were collected. On GD 21, all dams were euthanized and examined grossly. Gravid uterine weight was recorded to permit calculation of the adjusted maternal final body weight. The uterine contents were examined and described (number and status of implantation sites, and fetal assessment (viable, non-viable, location, sex, fetal weights, external alterations). There were no test substance-related maternal or developmental effects noted for any endpoint analyzed at any dose level. Based on these data, the dose levels selected for the current study were 30, 100, 300, and 1000 mg/kg/day.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (GD 6 - 20), once daily on GD 21

BODY WEIGHT: Yes
- Time schedule for examinations: once daily on GD 6 -21

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
- Time schedule for examinations: GD 6, 8, 10, 12, 14, 16, 18, 20 and 21

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: livers were weighed and saved for histopathological examination

OTHER: Gross external and a visceral examinations were performed. In the absence of any demonstrated test substance-related effects on either the gross findings or liver weights, microscopic examination of the livers and gross lesions was not considered necessary.
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
- Other: number of pregnant (%)
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

- Remarks: In addition, all live fetuses with malformations visible at external examination were examined for soft tissue alterations; decapitation of these fetuses for head examination was performed at the discretion of the study director or designee. The frozen heads of decapitated fetuses (fetuses that were decapitated prior to visceral examination, approximately half of the fetuses) were examined by a serial sectioning technique. The skeletal bodies of all the fetuses and the skulls of half the fetuses (fetuses that were not designated for head examination) were examined for alterations.
Statistics:
The level of significance selected was p < 0.05.
For an detailed overiew on statistics, please refer to table 1 in the "Any other information on materials and methods incl. tables" section.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: stained skin/fur - brown was observed in a single animal

100 mg/kg bw/day: wet fur was observed in a single animal

control, 30, 100 and 300 mg/kg bw/day: hair loss was observed in single animals at different time points.

All observations that were recorded were unremarkable and occurred infrequently and were thus not considered adverse.

For details please refer to table 2 in the “Any other information on results incl. tables” section.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
For details please refer to table 3 in the “Any other information on results incl. tables” section.

Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
100 mg/kg bw/day: liver discoloration was observed in one animal. This single event was considered incidental and non-adverse.
For details please refer to table 4 in the “Any other information on results incl. tables” section.

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):
For details please refer to table 5 in the “Any other information on results incl. tables” section.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
For details please refer to table 5 in the “Any other information on results incl. tables” section.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
For details please refer to table 5 in the “Any other information on results incl. tables” section.
Early or late resorptions:
no effects observed
Description (incidence and severity):
For details please refer to table 5 in the “Any other information on results incl. tables” section.
Dead fetuses:
no effects observed
Description (incidence and severity):
For details please refer to table 5 in the “Any other information on results incl. tables” section.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
For details please refer to table 5 in the “Any other information on results incl. tables” section.
Other effects:
not examined
Details on maternal toxic effects:
There were no test substance-related effects on reproductive outcome. The number of pregnant, the mean number of corpora lutea, implantation sites, resorptions (total, early and late), live/dead fetuses were comparable across all groups tested.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
For details please refer to table 5 in the “Any other information on results incl. tables” section.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
For details please refer to table 5 in the “Any other information on results incl. tables” section.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
For details please refer to table 5 in the “Any other information on results incl. tables” section.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
300 mg/kg bw/day: single incidences of protruding tongue (malformation), anopthalmia (malformation) and small eye bulge (gross finding) were observed, but considered an incidental finding and not considered adverse.
For details please refer to table 6 in the “Any other information on results incl. tables” section.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
300 mg/kg bw/day: single incidence of fused cervical arch (malformation) was observed, but considered an incidental finding and not considered adverse.
control, 30, 100, 300 and 1000 mg/kg bw/day: in all dose groups variations of the thoracic centrum and ribs were observed. In addition variations of the sternebrae were observed in all groups, exluding the 30 mg/kg bw/day dose group.
All observed variations were either incidental findings, not dose-related or occurrence did not differ significantly between dose groups and were thus not considered adverse.
For details please refer to table 6 in the “Any other information on results incl. tables” section.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
100 mg/kg bw/day: single incidence of discolored intestine (gross finding) was observed, but considered an incidental finding and thus not adverse.
30 mg/kg bw/day: single incidence of discolored liver (variation) was observed, but considered an incidental finding and thus not adverse.
For details please refer to table 6 in the “Any other information on results incl. tables” section.
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
There were no test substance-related effects on quantitative litter data. The number of live/dead fetuses, mean fetal weight and sex ratio were comparable across all groups tested. There were no test substance-related fetal malformations or variations observed at any dose level tested. The fetal alterations that were observed were unremarkable and occurred with low frequency across the dose levels tested.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 2: Summary of Maternal Clinical Observations

Dose group (mg/kg bw/day)

 

0

 

30

 

100

 

300

1000

Number of animals

 

25

 

25

 

25

 

25

25

Hair loss

Number of Observations

Number of animals

 

                       

 

 

4

1

 

 

17

3

 

 

16

3

 

 

3

3

 

 

Days from - to

16

19

12

21

15

21

19

21

 

Scheduled sacrifice

Number of Observations

Number of Animals

25

25

25

25

25

25

25

25

25

25

Days from - to

21

21

21

21

21

21

 21

21

 21

21

Stained skin/fur - brown – chin Number of Observations

Number of Animals

 

 

 

 

 

1

1

Days from - to

 

 

 

 

19

19

Wet fur

– chin/perinasal Number of Observations

Number of Animals

 

 

 

1

1

 

 

Days from - to

 

 

18

18

 

 

 

Table 3: Mean Final Body Weights, Absolute and Relative Organ Weights for Maternal Rats

Dose group (mg/kg bw/day)

0

30

100

300

1000

Final body weight

 

392.6

 

394.6

 

397.8

 

393.5

 

386.6

 

19.9(25)

21.8(25)

23.3(25)

24.2(25)

25.7(25)

Liver

Mean

15.40

15.04

15.49

15.71

14.72

Standard deviation (n)

1.70(24)

1.31(25)

1.66(25)

2.00(25)

1.38(24)

Liver/Final body weight x 100

 

3.922

 

3.816

 

3.891

 

3.994

 

3.813

Standard deviation (n)

0.350(24)

0.315(25)

0.294(25)

0.439(25)

0.345(24)

 

Table 4: Summary of Maternal Gross Observations

Dose group (mg/kg bw/day)

0

30

100

300

1000

Number of animals

25

25

25

25

25

Liver

No visible lesions

Discoloration

 

              

 

25

0

 

24a

0

 

23

1

 

23b

0

 

25

0

 

Whole Body

No visible lesions

 

25

 

 

24a

 

 

24

 

 

23b

 

 

25

 

 

a Maternal gross observations were inadvertently not recorded for Animal #204.

b Maternal gross observations were inadvertently not recorded for Animal #401 and #409.

 

Table 5: Reproductive Outcome

Dose group (mg/kg bw/day)

0

30

100

300

1000

Number of animals in group

25

25

25

25

25

Not Pregnant (%)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

Died/Killed

0

0

0

0

0

Survived to scheduled kill

0

0

0

0

0

Pregnant (%)

25(100.0)

25(100.0)

25(100.0)

25(100.0)

25(100.0)

Died/Killed/Aborted

0

0

0

0

0

with total resorption

0

0

0

0

0

with live fetus at scheduled kill

25

25

25

25

25

Corpora Lutea (Mean)

13.5

13.3

13.6

13.7

13.7

Standard Deviation (n)

3.1(25)

2.6(24)

3.0(25)

2.0(25)

2.2(25)

Implants (Mean)

11.7

12.2

11.9

12.3

12.3

Standard Deviation (n)

1.3(25)

2.2(25)

1.9(25)

1.7(25)

2.1(25)

Total Resorptions (Mean)

0.08

0.08

0.20

0.56

0.40

Standard Deviation (n)

0.28(25)

0.28(25)

0.50(25)

2.02(25)

0.65(25)

Early Resorptions (Mean)

0.08

0.08

0.20

0.56

0.40

Standard Deviation (n)

0.28(25)

0.28(25)

0.50(25)

2.02(25)

0.65(25)

Late Resorptions (Mean)

0.00~

0.00

0.00

0.00

0.00

Standard Deviation (n)

0.00(25)

0.00(25)

0.00(25)

0.00(25)

0.00(25)

Dead Fetuses (Mean)

0.0~

0.0

0.0

0.0

0.0

Standard Deviation (n)

0.0(25)

0.0(25)

0.0(25)

0.0(25)

0.0(25)

Live Fetuses (Mean)

11.6

12.2

11.7

11.7

11.9

Standard Deviation (n)

1.3(25)

2.2(25)

2.0(25)

2.7(25)

2.1(25)

Male Fetuses (Mean)

5.5

5.6

5.6

6.2

6.0

Standard Deviation (n)

1.8(25)

2.0(25)

2.3(25)

1.9(25)

2.1(25)

Female Fetuses (Mean)

6.1

6.6

6.2

5.6

5.8

Standard Deviation (n)

1.7(25)

2.1(25)

2.0(25)

2.0(25)

2.1(25)

Fetal Weight (Mean)

5.70

5.53

5.64

5.50

5.53

Standard Deviation (n)

0.24(25)

0.30(25)

0.25(25)

0.45(25)

0.27(25)

Male Weight (Mean)

5.89

5.70

5.77

5.67

5.66

Standard Deviation (n)

0.28(25)

0.30(25)

0.26(25)

0.41(25)

0.27(25)

Female Weight (Mean)

5.55

5.38

5.52

5.28

5.44

Standard Deviation (n)

0.26(25)

0.31(25)

0.28(25)

0.48(24)

0.32(25)

Sex Ratio (Mean)

0.47

0.47

0.47

0.54

0.51

Standard Deviation (n)

0.14(25)

0.15(25)

0.17(25)

0.16(25)

0.15(25)

~ next to control mean indicates no analyses were performed. Statistical analyses are only conducted on the total mean fetal weight; the means for males and females are presented for information only. Remarks: The incidence of pregnancy, maternal mortality, the total resorptions, and early deliveries/abortions were statistically analyzed using the Cochran-Armitage test. Live fetuses, dead fetuses, resorptions, corpora lutea, and implantations were analyzed by One-Way Analysis of Variance and Dunnett’s test. Fetal weight and sex ratio were analyzed using Analysis of Covariance and Dunnett-Hsu.

 

Table 6: Incidence of Fetal Malformations and Variations

Dose group (mg/kg bw/day)

0

30

100

300

1000

Total number of foetuses examined

290

304

293

293

297

External Defects: Number of Fetuses Examined

 

290

304

293

293

297

External Defects: Number of Litters Examined

25

25

25

25

25

Head

Tongue, Protruding (Malformation)

 

 

 

1 (0.3)

1 (4.0)

 

Head

Eye bulge, Small (Gross finding)

 

 

 

1 (0.3)

1 (4.0)

 

Head Defects: Number of fetuses examined

140

145

140

142

143

Head Defects: Number of litters examined

25

25

25

25

25

Head

Eye, Anophthalmia (Malformation)

 

 

 

1 (0.7)

1 (4.0)

 

Visceral Defects: Number of fetuses examined

140

145

140

142

143

Visceral Defects: Number of litters examined

25

25

25

25

25

Abdomen

Intestines, discolored (Gross finding)

 

 

1 (0.7)

1 (4.0)

 

 

Abdomen

Liver, discolored (Variation)

 

1 (0.7)

1 (4.0)

 

 

 

Skeletal - Head Defects: Number of fetuses examined

150

159

153

151

154

Skeletal - Head Defects: Number of litters examined

25

25

25

25

25

Skull

Frontal, Incomplete ossification (Variation)

 

 

5 (3.3)

1 (4.0)

 

 

Skull

Zygomatic, Incomplete ossification (Variation)

 

1 (0.7)

1 (4.0)

 

 

 

Skull

Interparietal, Incomplete ossification (Variation)

 

 

 

1 (0.7)

1 (4.0)

 

Skull

Supraoccipital, Incomplete ossification (Variation)

3 (2.0)

2 (8.0)

2 (1.3)

2 (8.0)

1 (0.7)

1 (4.0)

3 (2.0)

2 (8.0)

 

Skull

Parietal, Incomplete ossification (Variation)

 

1 (0.6)

1 (4.0)

1 (0.7)

1 (4.0)

2 (1.3)

2 (8.0)

 

Skeletal - Body Defects: Number of fetuses examined

290

304

293

293

297

Skeletal - Body Defects: Number of litters examined

25

25

25

25

25

Vertebrae

Cervical arch, Fused (Malformation)

 

 

 

1 (0.3)

1 (4.0)

 

Vertebrae

Thoracic centrum, Unossified (Variation)

 

 

 

1 (0.3)

1 (4.0)

 

Vertebrae

Thoracic centrum, Bipartite ossification (Variation)

6 (2.1)

6 (14.0)

1 (0.3)

1 (4.0)

2 (0.7)

2 (8.0)

1 (0.3)

1 (4.0)

8 (2.7)

6 (24.0)

Ribs

Rib, Short (Variation)

2 (0.7)

2 (8.0)

1 (0.3)

1 (4.0)

5 (1.7)

2 (8.0)

1 (0.3)

1 (4.0)

2 (0.7)

1 (4.0)

Ribs

Rib, Cervical rib (Variation)

3 (1.0)

2 (8.0)

3 (1.0)

1 (4.0)

4 (1.4)

2 (8.0)

1 (0.3)

1 (4.0)

 

Ribs

Rib, Full supernumerary rib (Variation)

3 (1.0)

1 (4.0)

 

 

 

 

Ribs

Rib, Thickened (Variation)

3 (1.0)

1 (4.0)

 

 

 

 

Ribs

Rib, Wavy (Variation)

4 (1.4)

1 (4.0)

 

1 (0.3)

1 (4.0)

 

 

Ribs

Rib, Short supernumerary (Variation)

2 (0.7)

2 (8.0)

2 (0.7)

2 (8.0)

2 (0.7)

2 (8.0)

 

2 (0.7)

1 (4.0)

Ribs

Rib, Extra ossification site (Variation)

19 (6.6)

10 (40.0)

13 (4.3)

6 (24.0)

10 (3.4)

9 (36.0)

 

9 (3.1)

4 (16.0)

5 (1.7)

5 (20.0)

Sternebrae

Unossified (Variation)

 

 

 

4 (1.4)

2 (8.0)

 

Sternebrae

Misaligned (Variation)

 

 

2 (0.7)

2 (8.0)

 

1 (0.3)

1 (4.0)

Sternebrae

Fused (Variation)

1 (0.3)

1 (4.0)

 

 

1 (0.3)

1 (4.0)

 

Upper line denotes number of affected fetuses

Lower line denotes number of affected litters

Figures in parenthesis denote percentage incidence

Calculated values do not include animals which either, were not pregnant, did not survive to the scheduled kill, had a total litter loss, aborted or are marked for exclusion

Applicant's summary and conclusion

Conclusions:
CLP: not classified