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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
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

Constituent 1
Reference substance name:
o-toluidine hydrochloride
IUPAC Name:
o-toluidine hydrochloride
Constituent 2
Chemical structure
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.