Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-02-20 to 2002-03-27
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
yes
Remarks:
source of test material not stated
GLP compliance:
yes
Type of assay:
other: Micronucleus Assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name as cited in the study report: 3-Methylbenzoic acid (MTA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot No. 1015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in a light-resistant container at room temperature
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- pulverisation to a fine powder in an agate mortar

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Remarks:
IGS
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS - Crj: CD(SD), IGS
- Source: Charles River Japan, Inc. (IGS refers to animals bred using the Charles River International Genetic Standardisation system)
- Age at study initiation (at administration): 7 weeks
- Weight at study initiation (at administration): 259 - 277 g (confirmation test: 266 - 289 g)
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing:
- Diet (ad libitum): pellet diet for experimental animals (MF, Oriental Yeast Co., Ltd.)
- Water (ad libitum): tap water, filtered through 5-µm filter and irradiated with UV light
- Acclimation period: 5 days
- Levels of contaminants were below acceptable upper limits specified at the test facility

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 15 %
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 1 w/v % MC (methylcellulose)
- Concentration of test material in vehicle: serial dilution: 200, 100, 50, 25 and 12.5 mg/mL (for usage in the main test and the confirmation test)
- Lot/batch no.: Metolose ® SM-100, Shin-Etsu Chemical, Inc., Lot no. 002658
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- suspended by addition of 1 w/v % methylcellulose
- adjusting a concentration of 200 mg/mL
- in an stepwise dilution procedure by adding 1 w/v % methylcellulose, suspensions of 100 mg/mL and 50 mg/mL were prepared
- in the confirmation test the test material was prepared as described previously, but diluted to a concentration of 50 mg/mL, 25 mg/mL and 12.5 mg/mL
- the dose solutions were prepared under yellow light
Duration of treatment / exposure:
2 days
Frequency of treatment:
twice at 24 hour intervals
Post exposure period:
24 hours (after the final administration)
Doses / concentrationsopen allclose all
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
confirmation tes
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
confirmation test
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 animals (male) per dose
Control animals:
yes, concurrent vehicle
yes, historical
Positive control(s):
cyclophosphamide (obtained from Sigma Chemical Company, Lot no. 86F-0101)
- Route of administration: intraperitoneally administered (once)
- Doses / concentrations: 10 mg/kg

Examinations

Tissues and cell types examined:
1000 erythrocytes were scored from each slide
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- doses were set according to a single dose toxicity study by the sponsor with LD50 >= 2000 mg/kg
- dose selection in the confirmation test: doses were set according to the main test which resulted in an unusual value in 1 of 5 animals of the low dose group (500 mg/kg)

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- the test substance and the negative control was administered by oral gavage twice at 24 hour intervals.
- the positive control was administered once intraperitoneally
- 24 hours after the last administration rats were sacrificed by exsanguination from the abdominal aorta under anesthesia (thiopental sodium) and femurs were dissected


DETAILS OF SLIDE PREPARATION:
- bone marrow cells were collected with phosphate buffered saline (PBS) and suspensions were centrifuged at 200 rpm for 5 min
- supernatant was transferred to 10 % neutral buffered formalin solution and centrifuged at 1000 rpm for 5 min
- cells were twice washed with 10 % neutral buffered formalin solution
- cells were resuspended in 10 % neutral buffered formalin solution
- suspension was stained with acridine orange and transferred to slides for observation

METHOD OF ANALYSIS:
- slides were observed and scored under blind condition with fluorescent microscope with B-2 excitation filter
- 1000 erythrocytes were scored from each slide in order to determine the ratio of polychromatic erythrocytes (PCEs) to the total erythrocytes (PCEs and normochromatic erythrocytes (NCEs))
- PCEs were further scored up to 2000 cells, the number of micronucleated PCEs (MNPCEs) in a slide were examined
- PCEs and NCEs were identified according to the method of Hayashi et al.*

*Reference:
Hayashi M, Sofuni T, Ishidate M Jr. (1983): An application of acridine orange fluorescent staining to the micronucleus test. Mutat. Res. 120, 241 - 247
Evaluation criteria:
When test substance induced a significant increase in the total number of micronucleated polychromatic erythrocytes with dose-dependency, the test is considered positive
Statistics:
- for the analysis of the percentage of polychromatic erythrocytes (PCEs): Student's t-test
- for the incidence of micronucleated PCEs (MNPCEs): tables of Kastenbaum and Bowman*
- statistics were conducted at the significance levels of 5 % and 1 %

*Reference:
Kastenbaum MA, Bowman KO (1970): Tables for determining the statistical significance of mutation frequencies. Mutat Res. 9, 527 - 549

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
- significant induction of micronuclei in the 500 mg/kg dose group: mean incidence of MNPCE = 0.68 ± 1.19

- Ratio of PCE/NCE (for Micronucleus assay):
- 500 mg/kg dose group: mean ratio PCE/NCE: 54.0 ± 1.0
- 1000 mg/kg dose group: mean ratio PCE/NCE: 53.5 ± 2.3
- 2000 mg/kg dose group: mean ratio PCE/NCE: 52.2 ± 2.6

RESULTS OF CONFIRMATION TEST
- Induction of micronuclei (for Micronucleus assay): no significant induction observed

- Ratio of PCE/NCE (for Micronucleus assay):
- 125 mg/kg dose group: mean ratio PCE/NCE: 50.4 ± 3.3
- 250 mg/kg dose group: mean ratio PCE/NCE: 49.1 ± 5.1
- 500 mg/kg dose group: mean ratio PCE/NCE: 50.5 ± 2.8

Please refer to the field 'Any other information on results incl. tables'

Any other information on results incl. tables

Further observation:

body weight: no effects observed

clinical signs: no effects observed

Table 1: Results of micronucleus test

Treatment group

Dosage (mg/kg) x times

Animal no.

Number of PCEs scored

MNPCE

PCE/(PCE+NCE)5

Number (total)

Incidence Mean

± SD

(%)

Mean

± SD

Negative control

0 x 21

1

2000

2

0.10

53.9

2

2000

3

0.15

55.2

3

2000

1

0.05

54.9

4

2000

2

0.10

56.0

5

2000

1

0.05

54.0

 

 

(9)

0.09 ± 0.04

54.8 ± 0.9

Test substance

500 x 21

1

2000

3

0.15

53.8

2

2000

3

0.15

55.5

3

2000

3

0.15

54.0

4

2000

3

0.15

52.7

5

2000

56

2.80

53.9

 

 

(68)3**

0.68 ± 1.19

54.0 ± 1.0

Test substance

1000 x 21

1

2000

2

0.10

50.9

2

2000

4

0.20

55.7

3

2000

1

0.05

55.6

4

2000

4

0.20

54.1

5

2000

5

0.25

51.2

 

 

(16)

0.16 ± 0.08

53.5 ± 2.3

Test substance

2000 x 21

1

2000

3

0.15

48.4

2

2000

1

0.05

55.0

3

2000

4

0.20

54.2

4

2000

4

0.20

52.0

5

2000

4

0.20

51.3

 

 

(16)

0.16 ± 0.07

52.2 ± 2.6

Positive control

10 x 12

1

2000

40

2.00

50.4

2

2000

35

1.75

53.6

3

2000

44

2.20

50.4

4

2000

62

3.10

48.3

5

2000

48

2.40

50.9

 

 

(229)3**

2.29 ± 0.51

50.7 ± 1.94##

PCE: polychromatic erythrocytes, MNPCE: micronucleated PCE, NCE: normochromatic erythrocytes

1twice administered by oral gavage at 24 hour interval

2once administered by intraperitoneal injection

3significantly different from the negative control (**p<0.01) by Kastenbaum & Bowman’s method

4significantly different from the negative control (##p<0.01) by Student’s t-test

51000 erythrocytes were scored

 

 

Table 2: Results of confirmation test

Treatment group

Dosage (mg/kg) x times

Animal no.

Number of PCEs scored

MNPCE

PCE/(PCE+NCE)5

Number (total)

Incidence Mean

± SD

(%)

Mean

± SD

Negative control

0 x 21

1

2000

1

0.05

57.7

2

2000

3

0.15

50.8

3

2000

2

0.10

53.9

4

2000

1

0.05

58.2

5

2000

1

0.05

52.9

 

 

(8)

0.08 ± 0.04

54.7 ± 3.2

Test substance

125 x 21

1

2000

2

0.10

46.5

2

2000

1

0.05

19.4

3

2000

1

0.05

52.3

4

2000

1

0.05

48.8

5

2000

3

0.15

55.0

 

 

(8)

0.08 ± 0.04

50.4 ± 3.3

Test substance

250 x 21

1

2000

1

0.05

51.9

2

2000

1

0.05

52.3

3

2000

2

0.10

48.9

4

2000

2

0.10

40.3

5

2000

4

0.20

51.9

 

 

(10)

0.10 ± 0.06

49.1 ± 5.1

Test substance

500 x 21

1

2000

3

0.15

 47.8

2

2000

2

0.10

50.5

3

2000

2

0.10

54.9

4

2000

2

0.10

48.2

5

2000

1

0.05

51.1

 

 

(10)

0.10 ± 0.04

50.5 ± 2.8

Positive control

10 x 12

1

2000

50

2.50

45.6

2

2000

28

1.40

43.1

3

2000

31

1.55

42.6

4

2000

44

2.20

37.3

5

2000

33

1.65

39.2

 

 

(186)3**

1.86 ± 0.47

41.6 ± 3.3

PCE: polychromatic erythrocytes, MNPCE: micronucleated PCE, NCE: normochromatic erythrocytes

1twice administered by oral gavage at 24 hour interval

2once administered by intraperitoneal injection

3significantly different from the negative control (**p<0.01) by Kastenbaum & Bowman’s method

4significantly different from the negative control (##p<0.01) by Student’s t-test

51000 erythrocytes were scored

 

Applicant's summary and conclusion

Conclusions:
Negative
The test item did not induce biologically relevant increases in micronucleated polychromatic erythrocytes in the bone marrow of rats when administered up to the limit dose of 2000mg/kg bw via oral route.