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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1978
Report date:
1978

Materials and methods

Test guidelineopen allclose all
Qualifier:
no guideline available
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
bone marrow sampling time 6h after last dose instead of 18h; highest dose tested caused deaths; verification of bone marrow exposure to the test substance not specified.
Principles of method if other than guideline:
REFERENCES: [1] Boller, K., Schmid W. (1970) Humangenetik 11, 35; [2] Heddle J.A. (1973) Mutation Res. 18, 187; [3] Matter B., Schmid W. (1971) Mutation Res. 12, 417; [4] Schmid W. (1973) Agents and Actions, 3, 77; [5] Schmid W. (1975) Mutation Res. 31, 9; [6] Von Ledebur M., Schmid W. (1973) Mutation Res. 19, 109.
GLP compliance:
not specified
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxy-4-(trifluoromethyl)benzoic acid
EC Number:
700-368-9
Cas Number:
328-90-5
Molecular formula:
C8H5F3O3
IUPAC Name:
2-hydroxy-4-(trifluoromethyl)benzoic acid
Test material form:
solid

Test animals

Species:
mouse
Strain:
CF-1
Remarks:
(CFLP, SPF)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Anglia Laboratory Animals (Alconbury, Huntingdon, UK)
- Age at study initiation: not specified
- Weight at study initiation: 19 - 23 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes, overnight
- Housing: each group of 5 mice was kept in a plastic disposable cage and maintained in a controlled environment.
- Diet: 'Grain Harvester' Anglia Laboratory Animal Diet, ad libitum.
- Water: tap water, ad libitum.
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC
- Humidity (%): 50 ± 5% RH

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 1% MC (methyl cellulose)
- Concentration of test material in vehicle: see experimental design in 'Any other information on materials and methods'.
- Dosing volume: 0.1 ml / 100 g bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was prepared as a suspension in 1% methylcellulose, by direct addition, using a high speed mixer.
Duration of treatment / exposure:
30h
Frequency of treatment:
2 equal doses separated by a 24-h interval
Post exposure period:
6h
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C;
- Route of administration: intraperitoneal injection
- Doses / concentrations: 14 mg/kg bw

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes (PCE) from bone marrow.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: preliminary studies, based on information provided, indicated that the LD50 of the test item is approximtely 400 mg/kg bw and that a top dosage of 1000 mg/kg bw would cause 1-2 deaths.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- Treatment: rats were administered the test item by oral gavage at total doses of 250, 500, and 1000 mg/kg bw, as two equal doses separated by 24h (both administered after overnight fasting) of 10 ml/kg-bw dose volume.

DETAILS OF SLIDE PREPARATION:
- Following the last dose, the animals were observed for a further 6h before killing and all mortalities and signs of malreaction during the experiment were recorded. The animals were killed by cervical dislocation, the femurs dissected out and bone marrow smears prepared. The smears were fixed in methanol, deffated in xylene and stained with Giemsa.

METHOD OF ANALYSIS:
- The stained smears were examined by light microscopy. 2000 polychromatic erythrocites (PCEs) per mouse were scored for the frequency of micronucleated cells.
Evaluation criteria:
In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 400 - 1400 mg/kg bw.
- Clinical signs of toxicity in test animals: No toxic reactions were observed up to 600 mg/kg bw. At doses of 800 - 1400 mg/kg bw, hypopnoea was observed. Mortality data is summarized in table 1.
- High dose with and without activation: a dose of 1000 mg/kg bw was chosen as the highest dose that would be tolerated without evidence of study-limiting toxicity, relative to the duration of the study period.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: No reactions were observed up to 500 mg/kg bw. At 1000 mg/kg bw, hypopnoea was observed, and there were three mortalities between 24 and 30 hours after administration of the second dose.
- Induction of micronuclei (for Micronucleus assay): No significant increase observed at any dose tested. The negative control group gave a mean count of 2.6 micronucleated cells, within the laboratory historical value range. The mean micronucleated cell counts for groups treated at 250 and 500 mg/kg bw were comparable with the concurrent control value. The mean micronucleated cell count for the group treated at 1000 mg/kg bw was 2.7, slightly higher than the concurrent control. However, individual counts were within the historical range for negative controls. Therefore, the value was not considered statistically significant. The positive control gave a mean count of 92.7 micronucleated cells.
- Ratio of PCE/NCE (for Micronucleus assay): not examined.

Any other information on results incl. tables

Table 1. Preliminary toxicity study: mortality data.

Phase

Group

Material

Total dosage

over 24 h

(mg/kg bw)

Mortality ratio

(no. deaths/no. dosed)

Period during which

deaths occurred (h)

Combined

0 – 8

8 – 24

24 – 30

I

1a

1% MC

-

0/2

0/2

0/4

 

 

 

2

UR1501

400

0/2

0/2

0/4

 

 

 

3

600

0/2

0/2

0/4

 

 

 

4

800

0/2

0/2

0/4

 

 

 

5

1000

0/2

0/2

0/4

 

 

 

6

1200

1/2

1/2

2/4

 

1

1

7

1400

1/2

1/2

2/4

 

 

2

II

1b

1% MC

-

0/5

0/5

0/10

 

 

 

8

UR1501

1000

1/5

2/5

3/10

 

 

3

9

1100

0/5

4/5

4/10

2

 

2

10

1200

5/5

3/5

8/10

6

 

2

11

1300

3/5

4/5

7/10

2

 

5

12

1400

5/5

3/5

8/10

5

 

3

Table 2. Main study: Group mean number of micronucleated cells per 2000 polychromatic erythrocytes per animal.

Group

Material

Total dosage

over 24 h

(mg/kg bw)

Period during which

deaths occurred (h)

Mean

Range

1

1% MC (vehicle control)

-

2.6

1 – 6

2

UR1501

250

2.2

1 – 5

3

500

2.1

0 – 6

4

1000

2.7

1 – 4

5

Mitomycin C (positive c.)

14

92.7

71 – 126

Table. Number of micronucleated cells (mn) per 2000 polychromatic erythrocytes – individual values.

 

Material

 

 

 

 

 

 

 

 

 

1% MC

UR1501

Mitomicyn C

Group

1

2

3

4

 

Dose (mg/kg)

-

250

500

1000

14

Sex

Animal

mn

Animal

mn

Animal

mn

Animal

mn

Animal

mn

1

1

6

3

11

2

16

D

21

81

2

6

7

1

12

4

17

2

22

77

3

2

8

1

13

1

18

2

23

126

4

3

9

5

14

2

19

4

24

93

5

2

10

2

15

1

20

1

25

118

26

2

31

3

36

2

41

4

46

77

27

2

32

2

37

2

42

D

47

71

28

4

33

2

38

6

43

D

48

94

29

1

34

1

38

1

44

4

49

102

30

3

35

2

40

0

45

2

50

88

D: animal died.

Table. Laboratory standard (historical) values in the period Feb 1972 – Feb 1978.

Negative control

No. of experiments

Total no. of

animals examined

Mean no. of micronucleated

cells counted per 2000 PCE

Range of individual counts

of micronucleated cells

per 2000 PCE per animal

81

783

1.65

0 – 6

 

Applicant's summary and conclusion

Conclusions:
The test item was not mutagenic under test conditions.
Executive summary:

An acute micronucleus study in bone marrow of CF-1 (CFLP, SPF) mice was performed for the test item, by a method similar to OECD 474 (non-GLP). Based on the results of a preliminary toxicity study were the LD50 was found to be around 400 mg/kg bw, five animals per sex per dose group were exposed to 250, 500 or 1000 mg/kg bw of test item in 1% methylcellulose by oral gavage, administered in two equal doses separated by a 24-hour resting period. Negative (vehicle, 1% aqueous methylcellulose) and positive (14 mg/kg bw Mitomicyn C by i.p. injection) controls were run in parallel. 6 hours after the last injection, the animals were killed by cervical dislocation, the femurs dissected out and and bone marrow spreads were prepared, air-dried, fixed using absolute methanol, defatted in xylene and stained with Giemsa. At termination, bone marrow analysis included assessment of the frequency of MN-PCE among 2,000 PCE per animal. All control values obtained were within the historical control range. No significant increase in the number of micronucleated PCE was observed at any dose tested, including the highest dose, which induced mortality. Since the results were clearly negative at all doses tested, the test item was not mutagenic under test conditions.

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