1,3-di-o-tolylguanidine

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:

2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

1,3-di-o-tolylguanidine

EC Number:

202-577-6

EC Name:

1,3-di-o-tolylguanidine

Cas Number:

97-39-2

Molecular formula:

C15H17N3

IUPAC Name:

1,3-di-o-tolylguanidine

Test animals

Species:

mouse

Strain:

ICR

Remarks:

Crlj:CD1 (ICR)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF, Charles River Laboratories Japan Inc
- Age at study initiation: 7 week-old
- Weight at study initiation: 26.8 31.6 g for the preliminary test, 25.7–29.5 g for the re-test of the preliminary test, and 29.1–33.3 g for the main test.
- Assigned to test groups randomly: yes, based on the body weights measured the day before the administration start date.
- Fasting period before study: no
- Housing: The animals were housed in bracket-type metal-mesh-floored cages (260W × 380D × 180H, mm). The number of animals housed per cage was no more than five during the quarantine and acclimation period, and no more than two after grouping.
- Diet (e.g. ad libitum): gamma-irradiated solid feed (CRF-1, Lot Nos. 060606 and 060706, Oriental Yeast Co., Ltd.) freely from a metal feeder.
- Water (e.g. ad libitum): Sapporo city municipal tap water (microfiltered) to drink freely using an automatic water supply device.
- Acclimation period: 5 or 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 10–15 times per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

0.5% CMC

Details on exposure:

Volume administration = 10 ml/kg

Duration of treatment / exposure:

single

Frequency of treatment:

two successive oral administration (24h apart)

Post exposure period:

24 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

Doses / concentrations 5

Dose / conc.:

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

No. of animals per sex per dose:

6 animals/ dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C, one single administration (intraperitoneal). Dose = 1 mg/kg

Examinations

Tissues and cell types examined:

bone marrow cells from femus

Details of tissue and slide preparation:

After the animals were euthanized by cervical dislocation 23–24 hours after the final administration, bone marrow cells from both femurs were washed out with foetal bovine serum (lot No.1299355, Gibco) and centrifugated (KR-702, Kubota Seisakusho K.K.) at 150 × g (1000 rpm) for five minutes. After excess serum was removed, a portion of the resuspended cell suspension was smeared onto a glass slide. Each slide was left to air-dry overnight at room temperature and then fixed with methanol (lot No. 801W1028, Kanto Chemical Co., Ltd.). Four slides were prepared for each animal.
After the slides were fixed with methanol, two slides for each animal were blinded by a person other than the observer.
After each of the selected slides was stained with 0.005% acridine orange staining solution (acridine orange, lot No. WAN0424, Wako Pure Chemical Industries, Ltd.), it was washed with 1/15 mol/L phosphate buffer solution (pH 6.8, lot No. A648, Mitsubishi Chemical Yatron Co., Ltd.), covered with a cover glass and sealed with enamel.
Slides were examined using a fluorescence microscope (BX50: BX-FLA, Olympus Optical Co., Ltd.) at a total magnification of 1000x. For each animal, 2000 immature red blood cells (1000 per specimenslide) were examined, and the frequency of occurrence of immature red blood cells with micronuclei among all immature red blood cells (incidence of micronucleus) was determined. In addition, 500 red blood cells (250 per slide) were examined for each animal, and the ratio of immature red blood cells among all red blood cells (ratio of immature red blood cells) was determined.

Evaluation criteria:

Results of the conditional binomial test were determined to be positive if the incidence of micronucleus in a test group was significantly higher than in the negative control group.

Statistics:

1) Body weight
The data for the negative control group and the test substance groups were tested for inter-group uniformity of dispersion by F-test. The results showed uniform dispersion, so pairs of groups were compared using Student’s t-test (two-sided). Two-sided significance levels were set at 5% and 1%.
2) Incidence of micronucleus
The incidence of micronucleus between the negative control group and the other groups (including the positive control group) was compared using a conditional binomial test (test based on the numerical tables of Kastenbaum and Bowman). The significance levels for the test were set at 5% and 1% on the upper side.
3) Ratio of immature red blood cells
The data for the negative control group and the test substance groups or the positive control group were tested for inter-group uniformity of dispersion by F-test. The results showed uniform dispersion, so pairs of groups were compared using Student’s t-test (two-sided). Two-sided significance levels were set at 5% and 1%.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

mortalities at 40 and 50 mg/kg

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Mortality : No mortality at 20.5, 25.6, 32 mg/kd/d, in negative and in positive controls.
2/6 died animal at 40 mg/kg within 1-6hr after the first exposure. 1/6 died animal at 50 mg/kg within 1-6hr after the first exposure.
Clinical signs : Absence of effects at all doses.
No change of bodyweight was observed.

Genotoxicity : no statistically significant results obtained for treated group when compared to the control vehicle group. Positive and statistically results were obtained in the positive control group.

Applicant's summary and conclusion

Conclusions:

Based on these results, 1,3-di-o-tolyguanidine was judged not to be clastogenic in vivo under the conditions of the main test.

Executive summary:

In order to investigate the clastogenicity of 1,3-di-o-tolyguanidine in vivo, a micronucleus test was performed using male mice. In test substance groups, on the basis of the results of preliminary testing (performed twice, including re-test), the test substance was administered in two oral administrations, with an interval of approximately 24 hours, at 20.5, 25.6, 32, 40, and 50 mg/kg/day. In a negative control group, a 0.5 w/v% carboxymethylcellulose sodium solution was administered in the same manner as in the test substance groups. In a positive control group, 1 mg/kg of mitomycin C was administered once intraperitoneally. Bone marrow smear specimens were prepared 23 to 24 hours after the second administration in each group, and the following results were obtained.

 

In observation of the general condition of the animals, no abnormal symptoms were seen in the negative control group or in the 20.5, 25.6, and 32 mg/kg/day test substance groups. Two of six animals in the 40 mg/kg/day group and one of six animals in the 50 mg/kg/day group died after the first administration.

Mean values for the body weights of the animals in the test substance groups were at the same level as values from before the first administration, and no clear difference from the negative control group was seen either.

The incidence rate of the count of immature red blood cells with micronuclei relative to total red blood cell count (incidence rate of micronucleus) was 0.17 ± 0.06% (mean ± S.D., n = 5) in the negative control group, while in the 20.5, 25.6, 32, 40, and 50 mg/kg/day test substance groups, it was 0.10 ± 0.07% (n= 5), 0.13 ± 0.10% (n = 5), 0.17 ± 0.10% (n = 5), 0.21 ± 0.13% (n = 4), and 0.22 ± 0.07% (n = 5) respectively, with no statistically significant difference. So the test substance did not show any micronucleus induction.

The ratio of immature red blood cells to all red blood cells was 46.3 ± 4.8% (average value ± S.D., n = 5) in the negative control group, while in the test substance 20.5, 25.6, 32, 40, and 50 mg/kg/day groups, it was 50.0 ± 6.4% (n = 5), 47.1 ± 5.1% (n = 5), 47.7 ± 6.5% (n = 5), 46.7 ± 1.4% (n = 4), and 41.2 ± 4.1% (n = 5) respectively, with no statistically significant difference. So the test substance did not show any myelotoxicity.

In the positive control group, the incidence of micronucleus was 3.00 ± 0.50% (n = 5), a statistically significantly higher value than the negative control group was observed, indicating that the test has been conducted appropriately.

Based on these results, 1,3-di-o-tolyguanidine was judged not to be clastogenic in vivo under the conditions of the main test.