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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Jul - 20 Oct 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: UKEMS Guidelines (1990)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspects of Regulatory Tests (1995)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
446-630-3
EC Name:
-
Cas Number:
181587-01-9
Molecular formula:
C13H9Cl2F3N4OS
IUPAC Name:
5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-(ethanesulfinyl)-1H-pyrazole-3-carbonitrile
Details on test material:
- Name of test material (as cited in study report): RPA 107382
- Analytical purity: 93%
- Lot/batch No.: CDR 9706

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd, Margate, UK
- Age at study initiation: 35 days
- Weight at study initiation: 25-32 g (males), 21-27 (females)
- Assigned to test groups randomly: yes, under following basis: male and female mice were randomised to groups of five using a system of randomly generated numbers.
- Housing: animals were housed in groups of no more than four animals of the same sex in appropriate caging.
- Diet: diet (Special Diets Services Ltd, RM1.[E].SQC.), ad libitum
- Water: mains water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24
- Humidity (%): 45-65
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 0.5% methyl cellulose
- Concentration of test material in vehicle: 25, 50 and 100 mg/mL for dose levels of 500, 1000 and 2000 mg/kg bw/day, respectively
- Amount of vehicle: 20 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: dosing preparations were made by suspending the test substance (with homogenisation) in 0.5% methyl cellulose (0.5% MC) to give the top concentrations of 25, 50 and 100 mg/mL for dose levels of 500, 1000 and 2000 mg/kg bw/day, respectively, and dilutions were made using 0.5% MC. The test article preparations were protected from light, maintained as an even suspension (by multiple inversion) and used within 6.5 h of initial formulation (following analysis of achieved concentration).
Duration of treatment / exposure:
not applicable
Frequency of treatment:
Range-finding study and main study: single administration
Post exposure period:
Range-finding study: 4 days after treatment
Main study: 24, 48 and 72 h after treatment with the test substance; 24 h after treatment with the positive control CPA
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Range-finding study: 3
Main study: 5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw (2 mg/mL in saline)

Examinations

Tissues and cell types examined:
Tissue: bone marrow
Cell type: bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: a range finding study was performed to find the maximum tolerated dose. The test article in 0.5% CM was administered as a single dose to groups of three male and three female mice at 2000 mg/kg/day via oral gavage. Observations were made over a 4-day period following administration and signs of toxicity and body weight recorded. These data were used to obtain a maximum acceptable dose (2000 mg/kg/day) which was used as the top dose in the main study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): sampling was performed 24, 48 and 72 h after single administration of the test substance and 24 h after single administration of the positive control CPA.

DETAILS OF SLIDE PREPARATION: slides were fixed with methanol and then stained with filtered Giemsa solution (1:6 (v/v) diluted in distilled water) for 10 min.

METHOD OF ANALYSIS: slides were microscopically analysed and relative proportions of polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were determined until a total of at least 1000 cells (PCE plus NCE) had been analysed. PCE/NCE ratios were examined to see if there was any decrease in groups of treated animals that could be taken as evidence of bone marrow toxicity. Counting continued (but of PCE only) until at least 2000 PCE had been observed. All PCE containing micronuclei observed during these two phases of counting were recorded. The vernier coordinates of all cells containing micronuclei were recorded to a maximum of six per 2000 cells scored.
Evaluation criteria:
The test article was to be considered as positive in this assay if:
1) a statistically significant increase in the frequency of micronucleated PCE occurred at least at one dose, at one sampling time, was observed and
2) the frequency of micronucleated PCE at such a point exceeded the historical vehicle control range.
If statistically insignificant increases in frequencies of micronucleated PCE at one dose, at one sampling time in conjunction with either 1 or 2 of the above at the other sampling time were observed, additional testing or analysis was needed.
Statistics:
The ratio of PCE/NCE for each animal and the mean for each group was calculated and the frequency of micronucleated PCE/1000 PCE was determined. The individual and group mean frequencies of micronucleated PCE/1000 cells (± standard deviation) were also determined. The group mean frequencies of micronucleated PCE in vehicle control animals were compared with historical negative control ranges to determine whether or not the assay was acceptable. For each group, interindividual variation in the numbers of micronucleated PCE was estimated by means of a heterogeneity χ2 test. The numbers of micronucleated PCE in each treated group (males and females, separately and combined) were then compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine χ2. Probability values of p < 0.05 were to be accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
1000 mg/kg bw/day: mortalities were observed in 1/5 females 24 h after treatment and in 1/5 males 48 h after treatment; 2000 mg/kg bw/day: mortalities were observed in 1/5 females 24 h after treatment and in 2/5 females 48 h after treatment
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: based on the results of the range finding study, dose levels of 500, 1000 and 2000 mg/kg bw were selected for the main study.
- Solubility: the test substance was well soluble in 0.5% CM.
- Clinical signs of toxicity in test animals: abnormal gait was exhibited by both sexes between 2 and 4 hours after administration. No other clinical signs or loss of body weight were observed.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: the test substance did not induce an increase in the frequency of micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 2000 mg/kg bw. Group mean frequencies of micronucleated PCE were similar to those seen in vehicle control groups and were not significantly different after statistical analysis. All micronuclei frequencies fell within the historical vehicle control range.
- Ratio of PCE/NCE: groups of mice treated with the test substance exhibited PCE/NCE ratios which were similar to those treated with the vehicle at all sampling times.
- Appropriateness of dose levels and route: dose levels and route of exposure were appropriate, since at least eight animals (males plus females) out of each group at each kill time were available for analysis. Thus, the acceptance criteria for the micronucleus assay were fulfilled (see “Any other information on materials and methods incl. tables”).
- Statistical evaluation: the numbers of micronucleated PCE in each treated group (males and females, separately and combined) were then compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine χ2. Probability values of p < 0.05 were to be accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships. No statistically significant differences were observed in the frequency of micronucleated polychromatic erythrocytes between treated and control animals.

Any other information on results incl. tables

VALIDITY OF THE STUDY

The acceptance criteria outlined under “Any other information on materials and methods incl. tables” were fulfilled, as shown by the following results:

1) in most cases, the heterogeneity χ2 test provided evidence of acceptable variability in the number of micronucleated PCE between animals within each group. Heterogeneity of marginal significance (p ≤ 0.05) was seen among animals treated with the vehicle and sampled at 48 hours and was attributable to one mouse with 4 micronucleated PCE/2000 cells. Insofar as such a frequency fell within the historical vehicle control range and has previously been seen among control animals, and other animals in the group had low frequencies of cells with micronuclei, it was considered to be of no biological significance.

2) The incidence of micronucleated PCE in vehicle control groups fell within the historical vehicle control range.

3) At least eight animals (males plus females) out of each group at each kill time were available for analysis.

4) The positive control chemical (CPA) induced a statistically significant increase in the frequency of micronucleated PCE.

RESULTS OF THE MAIN ASSAY

Table 1. Results of the in vivo micronucleus assay in male animals

Male treatment group

Mean ratio PCEs / NCEs
at sampling time

Mean frequency of micronucleated PCEs (per 1000 cells) at sampling time

Group

Number of animals

Dose [mL/kg]

24 h

48 h

72 h

24 h

48 h

72 h

Vehicle control (0.5% methyl cellulose)

5

20

1.21

0.79

1.16

0.30

0.00

0.25

Positive control (CPA)

5

20

0.98

n.d.

n.d.

12.50

n.d.

n.d.

500 mg/kg bw

5

20

0.96

0.65

0.96

0.69

0.30

0.20

1000 mg/kg bw

5

20

1.15

0.61

0.80

0.30

0.25

0.30

2000 mg/kg bw

5

20

1.38

0.71

0.97

0.10

0.20

0.10

n.d. = not determined; CPA = cyclophosphamide

Table 2. Results of the in vivo micronucleus assay in female animals.

Female treatment group

Mean ratio PCEs / NCEs
at sampling time

Mean frequency of micronucleated PCEs (per 1000 cells) at sampling time

Group

Number of animals

Dose [mL/kg]

24 h

48 h

72 h

24 h

48 h

72 h

Vehicle control (0.5% methyl cellulose)

5

20

0.99

0.81

0.89

0.70

0.79

0.50

Positive control (CPA)

5

20

0.80

n.d.

n.d.

12.18

n.d.

n.d.

500 mg/kg bw

5

20

1.14

0.98

0.90

0.60

0.70

0.10

1000 mg/kg bw

5

20

1.14

0.80

1.04

0.12

0.20

0.20

2000 mg/kg bw

5

20

0.98

0.76

0.82

0.25

0.12

0.25

n.d. = not determined; CPA = cyclophosphamide

Table 3. Results of the in vivo micronucleus assay (per treatment group)

Treatment group

Dose [mg(kg bw]

Sampling time [h]

Mean frequency of PCE with MN (per 1000 cells) (± SD)

PCE/NCE ratio

Vehicle control

0

24

0.50 ± 0.6

1.10

48

0.44 ± 0.7

0.80

72

0.39 ± 0.3

1.03

Test substance

500

24

0.64 ± 0.6

1.05

48

0.50 ± 0.5

0.82

72

0.15 ± 0.2

0.93

Test substance

1000

24

0.22 ± 0.4

1.15

48

0.22 ± 0.3

0.71

72

0.25 ± 0.4

0.92

Test substance

2000

24

0.17 ± 0.3

1.18

48

0.17 ± 0.3

0.74

72

0.17 ± 0.4

0.90

Positive control (CPA)

40

24

12.34 ± 3.7*

0.89

CPA = cyclophosphamid; MN = Micronuclei; *p ≤ 0.001

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative