Barium m-toluate

Genetic toxicity: in vivo

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

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

Test material information

Constituent 1

Reference substance name:

m-toluic acid

EC Number:

202-723-9

EC Name:

m-toluic acid

Cas Number:

99-04-7

Molecular formula:

C8H8O2

IUPAC Name:

3-methylbenzoic acid

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 or test system 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 / concentrations

Doses / concentrations 1

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Remarks:

confirmation tes

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

confirmation test

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Doses / concentrations 5

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 2^1		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 2^1		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 2^1		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 2^1		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 1^2		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 ± 1.9^4##

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 2^1		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 2^1		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 2^1		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 2^1		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 1^2		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.