3-chloro-o-toluidine

Genetic toxicity: in vivo

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:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted under GLP and according to guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

3-chloro-o-toluidine

EC Number:

201-756-6

EC Name:

3-chloro-o-toluidine

Cas Number:

87-60-5

Molecular formula:

C7H8ClN

IUPAC Name:

3-chloro-2-methylaniline

Details on test material:

- Name: 3-Chlor-2-methylanilin
- Code: Hoe CG 0221 OD ZD99 0001
- Other names: 6-Chlor-2-toluidin
- Batch No.: Straßentankzug/2 (Riedel), March 1990
- Purity: 98.8 %
- Certificate of analysis: No. 04877, dated January 10th, 1991, Analytisches Laboratorium, Dr. R. Fischbach
- Stability in solvent: stable for 5 hours , Analytisches Laboratorium, Toxikologie-Analytik, Dr. H .-J . Pletsch, dated November 18th, 199 3
- Storage stability: stable until December 1995, see certificate of analysis
- Storage conditions: dark at approximately 20°C
- Appearance: light liquid

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: 8 weeks
- Weight at study initiation: males 30-38 g; females 23-33 g.
- Assigned to test groups randomly: yes
- Housing: in air-conditionned rooms in Macrolon cages (Type 3), on softwood granulate in groups of 5 animals
- Diet: rat/mice diet Altromin 1324 (Altromin-GmbH, Lage/Lippe), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 50 +/- 20
- Photoperiod (hrs dark / hrs light): 12 h/12 h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

On the day of the experiment the test substance was dissolved in sesame oil at a appropriate concentration.

Duration of treatment / exposure:

Single gavage.

Frequency of treatment:

Once

Post exposure period:

12; 24 and 48 h after administartion, the animals were sacrificed.

Doses / concentrations

Remarks:

Doses / Concentrations:
800 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes

Positive control(s):

Endoxan was used as positive control and administrated at a dose of 50 mg/kg bw.

Examinations

Tissues and cell types examined:

Erythrocytes from bone marrow

Details of tissue and slide preparation:

In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 12, 24 or 48 hours after application. For each animal, about 3 mL foetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at approx. 1200 rpm and almost all the supematant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number and air-dried for about 12 hours.

Evaluation criteria:

1000 Polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation were coded to ensure that the group which they belong to remained unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically.

A Wilcoxon-Test (one-sided) was done to check the validity of the study by comparing the number of polychromatic erythrocytes with micronuclei in the positive control with the negative control. A Wilcoxon-Test (one-sided) was done for each measurement group (12h, 24h, 48h) and for polychromatic and normochromatic erythrocytes. These tests were performed sequentially with a multiple level of significance of 5%.

The presupposition to make any of the following comparisons is a difference between the positive control and the negative control (24h). This was tested with a Wilcoxon-Test (two-sided) with 5 %-level of significance. Wilcoxon-Tests (two-sided) were performed sequentially for the ratio of polychromatic erythrocytes for each measurement group (12h, 24h, 48h) at a multiple level of significance of 5 %. Actual data were also compared with historical controls.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

uncoordinated gait, forward crawling, prone position, palpebreal fissure narrow, reduced placing reflex, spontaneous activity decreased, coat bristing, cyanosis.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

						Erythrocytes		Poly		Erythrocytes with micronuclei
Sex	Dose [mg/kg bw]	Sample Time	Number of animals	Poly mean	normo mean	Mean	P/N, SD	NO	%	(Mean) SD	Mut. I.	NO	%	SD	Mut. I.
pooled	control	12 h	10	1000	1000	0.9	0.16	0.7	0.1	0.07	1	0.3	0	0.05	1
	800	12 h	10	1000	1000	1	0.2	1.2	0.1	0.08	1.7	0.6	0.1	0.07	2
pooled	control	24 h	10	1000	1000	0.7	0.13	0.8	0.1	0.08	1	0.5	0.1	0.07	1
	800	24 h	10	1000	1000	0.7	0.08	0.09	0.1	0.09	1.6	0.3	0	0.05	0.6
	p. control	24 h	10	1000	1000	0.9	0.19	1.11	3.3	1.11*	40.8	1.3	0.1	0.09	2.6
pooled	control	48 h	10	1000	1000	0.8	0.15	1	0.1	0.08	1	0.9	0.1	0.1	1
	800	48 h	10	1000	1000	0.9	0.11	1	0.1	0.08	1	0.6	0.1	0.1	0.7
Mut. I. = mutagenic index = erythrocytes with micronuclei in dose group / erythrocytes with micronuclei in control
Control = vehicle
p. control = positive control = endoxan
* = significantly different from control (p < 0.05)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative