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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented publication which meets basic scientific principles and with acceptable restrictions (individual animals data not given, no data on GLP status and substance purity)

Data source

Reference

Reference Type:

publication

Title:

Assays of Three Carcinogen/Non-Carcinogen Chemical Pairs for In Vivo Induction of Chromosome Aberrations, Sister Chromatid Exchanges and Micronuclei

Author:

McFee A.F, Jauhar P. P., Lowe K. W., MacGregor J.T. and Wehr C.M.

Year:

1989

Bibliographic source:

Environmental and Molecular Mutagenesis 14:207-220

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

- only one sex was employed

GLP compliance:

not specified

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

o-toluidine hydrochloride

IUPAC Name:

o-toluidine hydrochloride

Constituent 2

Reference substance name:

o-toluidinium chloride

EC Number:

211-252-8

EC Name:

o-toluidinium chloride

Cas Number:

636-21-5

Molecular formula:

C7H9N.ClH

IUPAC Name:

2-methylanilinium chloride

Test material form:

other: solid

Details on test material:

Name: o-toluidine hydrochloride
Purity: no data

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: 10-16 weeks
- Weight at study initiation: 20-30g (weight variation of ± 2g within each dose group)
- Housing: under conditions approved by the association for laboratory animal care
- Diet: ad libitum
- Water: ad libitum

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Dimethyl sulfoxide (DMSO)

Details on exposure:

Before study was started, the maximum tolerated dose (MTD) for the test compound in the respective solvent was initially established, using the criteria described by McFee 1989 or MacGregor et al. 1987). Three dose levels of the chemical were then utilized for the test (MTD, 0.5 MTD, and 0.25 MTD).

McFee AF, Lowe KW, San Sebastian IR (1983): Improved sister-chromatid differentiation using pariffin-coated bromodeoxyuridine tablets in mice. Mutat Res I 19:83-88.
MacGregor IT. Heddle JA. Hite M. Margolin BH. Ramel C. Salamone MF. Tice RR. Wild D(1987): Guidelines for the conduct of micronucleus assays in mammalian bone marrow erythrocytes. Mutat Res 189:103-112.

Duration of treatment / exposure:

24h, 48h, 72h

Frequency of treatment:

single application

Post exposure period:

no

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 75, 150, 300 mg/kg bw
Basis:
other: MTD, 1/ MTD, 1/4 MTD

No. of animals per sex per dose:

8 animals/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

7,12-dimethylbenz[a]anthracene (DMBA) in DMSO

Examinations

Tissues and cell types examined:

After sacrifice of animals marrow cells from a single femur of each animal were obtained by flushing with fetal calf serum

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
Marrow cells were flushed from a single femur of each animal with serum calf serum. Smears were air dried, fixed in absolute methanol and stained
with Hoechst 33258/pyronin Y using Sorensen's M/15 phosphate buffer.

Statistics:

Statistically analysis by the one-tail trend test of Margolin et al. 1986, and individual group means were further compared by a pairwise t-test utilizing the appropriate Bonferoni correction for the number of pairs of means examined.

Margolin BH. Resnick MA. Rimpo Y, Archer P. Galloway SM, Bloon AD. Zeiger E (1986): Statistical analysis for in vitro cytogenetic assays using Chinese hamster ovary cells. Environ Mutagen 8 183-204.
Kastenbaum-Bowmann tables

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CLINICAL OBSERVATIONS:
Injections of 300 mg/kg bw had a very pronounced sedative effect; within 20-30 min after injection the animals were totally immobile and showed minimal response to physical stimulus.
There was no indication of a significant increase in the number of micronucleated polychromatic erythrocytes in mice injected with the test substance. Although the percentage of marrow erythrocytes showing polychromatic staining reactions fluctuated, there was no apparent pattern that would suggest a consistent effect of the test compound in their maturation process (see table below)

Any other information on results incl. tables

Frequency of micronucleated polychromatic erythrocytes

	Micronucleated PCEs/1000 PCE			Percent PCE
Treatment mg/kg	24h	48h	72h	24h	48h	72h
75	2.39 ± 0.54	1.95 ± 0.37	0.56 ± 0.29*	56.8 ± 2.5	63.3 ±  3.0	67.5
150	1.57±0.40	2.28 ± 0.60	2.06 ± 0.69	48.4 ±2.0	37.5 ± 2.9	58.8
300	3.02 ± 0.43	2.45 ± 1.0  2.00 ± 0.42	2.52 ± 0.44	53.6 ±1.4	49.3 ±4.2 41.4 ±3.8	53.6
Trend P value	0.1554	0.3117	0.2490
DMSO 5 ml/kg	2.16 ± 0.50 2.25 ± 0.45	2.16 ± 0.47	2.65 ± 0.44	68.1 ± 3.1 54.5 ± 4.5	66.6 ± 3.6	61.3
DMBA 30 mg/kg	7.17 ± 1.75**	18.37   ± 2.09	(-)	56.4 ± 4.4	35.4 ± 4.7
P value	< 0.0001	< 0.0001

8 animals were evaluated except for (*) with 7 animals evaluated and ** with 5 animals evaluated

(-) PCE production suppressed in 4/5 mice. 1 mortality

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative

Executive summary:

In a MNT equivalent to OECD TG474 (only male mice used) o-Toluidine Hydrolchloride dissolved in DMSO was given as single i.p. injections to 8 B6C3F1 mice per dose (300,150, 75 mg/kg bw being MTD, 1/2 MTD, 1/4 MTD). Solvent/negative control and positive controls were included. Injections of 300 mg/kg bw had a very pronounced sedative effect; within 20-30 min after injection the animals were totally immobile and showed minimal response to physical stimulus. Sampling of cells from a single femur was done 24, 48, and 72 hours post application. o-toluidine hydrochloride yielded negative results.