6-Amino-5-chloro-2-cyclopyrimidine-4-carboxylic acid

Developmental toxicity / teratogenicity

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

Test material information

1

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 / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 4

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	24^a 0	23 1	23^b 0	25 0
Whole Body No visible lesions	25	24^a	24	23^b	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