N,N-dimethyl-p-toluidine

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

The study contains experimental data of the registered substance.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: As below explaned

Principles of method if other than guideline:

The ability of the registered substance to induce micronuclei in blood erythrocytes was tested in B6C3F1 mice after 14 weeks of oral administration.

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

N,N,4-Trimethylbenzenamine

Cas Number:

99-97-8

Molecular formula:

C9H13N

IUPAC Name:

N,N,4-Trimethylbenzenamine

Test material form:

liquid

Specific details on test material used for the study:

Purity: >99%

Test animals

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

Mice were obtained from Taconic Farms, Inc. (Germantown, NY). On receipt, the mice were 4 to 5 weeks old. Animals were quarantined for 11-14 days and were 5 to 7 weeks old on the first day of the study.

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

Groups of 10 male and 10 female mice received N,N-dimethyl-p-toluidine in corn oil by gavage, 5 days per week for 14 weeks.

Duration of treatment / exposure:

3 months (14 weeks)

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0

Remarks:

Vehicle control (corn oil)

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

60 mg/kg bw/day

Doses / concentrations 5

Dose / conc.:

125 mg/kg bw/day

No. of animals per sex per dose:

5 mice/sex/dose

Control animals:

yes, concurrent vehicle

Examinations

Tissues and cell types examined:

Blood erythrocytes

Details of tissue and slide preparation:

Peripheral blood samples were obtained from male and female mice at the end of the 3-month gavage study. Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs, mature erythrocytes) per animal. In addition, the percentage of polychromatic erythrocytes (PCEs, reticulocytes) among a population of 1,000 erythrocytes in the peripheral blood was scored for each dose group as a measure of bone marrow toxicity.

Evaluation criteria:

In the slide-based micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups.

Statistics:

The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over dose groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each dosed group and the control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

No significant increases in the frequencies of micronucleated erythrocytes, an indicator of chromosomal damage, were observed in peripheral blood of male or female B6C3F1/N mice treated with 15 to 125 mg/kg per day test substance by gavage for 3 months. No significant alterations in the percentage of circulating reticulocytes were observed, suggesting that the test substance did not induce bone marrow toxicity over the dose range tested.

Applicant's summary and conclusion

Conclusions:

The registered substance, N, N-Dimethyl-p-toluidine (CAS number: 99-97-8), did not induce the formation of micronucleated erythrocytes, as an indicator of chromosomal damage, in peripheral blood erythrocytes of male or female B6C3F1/N mice treated with 15 to 125 mg/kg per day test substance by gavage for 3 months.

Executive summary:

The ability of the registered substance, ,N, N-Dimethyl-p-toluidine (CAS number: 99-97-8), to induce micronuclei in blood erythrocytes was tested in B6C3F1 mice after 14 weeks of oral administration.Groups of 10 male and 10 female mice receivedthe test substancein corn oil by gavage at doses of 15, 30, 60, 125, or 250 mg/kg, 5 days per week for 14 weeks. Vehicle control animals received the corn oil vehicle alone. Feed and water were available ad libitum. The animals were weighed and clinical findings were recorded at study start, weekly thereafter, and at study termination.Mortality occurred at 125 mg/kg; three males and two females administered died before the end of the study. The final mean body weight of 125 mg/kg males and the mean body weight gains of 125 mg/kg males and females were significantly less than those of the vehicle controls.Clinical findings associated with administration ofthe test substanceincluded abnormal breathing, thinness, lethargy, cyanosis, and ruffled fur in 125 and 250 mg/kg males and females.At the end of theadministrating period (3months),peripheral blood samples were obtained from male and female mice. Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange.Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs, mature erythrocytes) per animal. In addition, the percentage of polychromatic erythrocytes (PCEs, reticulocytes) among a population of 1000 erythrocytes in the peripheral blood was scored for each dose group as a measure of bone marrow toxicity.The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over dose groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each dosed group and the control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. In the slide-based micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups.Results:No significant increases in the frequencies of micronucleated erythrocytes were observed in peripheral blooderythrocytesof male or female B6C3F1/N mice treated with 15 to 125 mg/kg/day test substanceby gavage for 3 months.No significant alterations in the percentage of circulating reticulocytes were observed, suggesting thatthe test substancedid not induce bone marrow toxicity over the dose range tested.Conclusion:The test substance did not inducechromosomal damagein peripheral blooderythrocytesof male or female B6C3F1/N mice treatedup to125 mg/kg/dayby gavage for 3 months.