1,3-di-o-tolylguanidine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

not specified

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration 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

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

DMSO was added to the cell suspension at a ratio of 10%, and the sample cells stored under liquid nitrogen conditions were returned to the medium, and used until the passage number reached four after thawing.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Without S9 mix (6 hr short-term treatment): 0, 75, 150, 300, 450, 600 µg/mL
With S9 mix (6 hr short-term treatment): 0, 75, 150, 300, 450, 600 µg/mL
With S9 mix (6 hr short-term treatment :Confirmation test): 0, 400, 600, 800 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The test substance is not soluble in water, and since the results of the preliminary study revealed that it is soluble in DMSO, the solvent used was DMSO.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: (-S9 mix) 1-Methyl-3-nitro-1-nitrosoguanidine, (+S9 mix) 3,4-Benzo[a]pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
The Eagle-MEM powdered medium was prepared according to the standard method, and inactivated (56°C, heat treated for 30 minutes) fetal bovine serum was added at a ratio of 10% for use.
Using a CO2 incubator, test cells were incubated under heated conditions of a CO2 concentration of 5%, with air of 95% and a temperature at 37°C.

DURATION
5 mL of the media containing 4x103 / mL of cells was added to a round plastic Petri dish (Becton Dickinson Co.) with a diameter of 6 cm. Three days after incubation started, treatment was conducted according to the following method. For incubation, four Petri dishes were used for each dose and two of these were used for preparation of the stained samples and the remaining two were used to measure the cell growth inhibition rate. For the positive control group, measurement of the cell growth rate was not performed, and two Petri dishes were used for preparation of the stained samples.
For the short term treatment without the S9 mix, the media was removed with 3 mL remaining in each Petri dish, and 0.015 mL of DMSO, the test substance sample solution and the MNNG sample solution were added to the Petri dishes for incubation. Additionally, with the S9 mix, the media was removed with 2.5 mL remaining in each Petri dish, and 0.5 mL of the S9 mix was added, and then 0.015 mL each of the DMSO, test substance sample solution and B[a]P sample solution was added to the Petri dishes for incubation. Both with and without the S9 mix, the media was removed 6 hours after incubation, the cell surfaces were washed one time with fresh media, and 5 mL of new media was added for 18 hours of incubation.

SPINDLE INHIBITOR (cytogenetic assays): colcemid

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY: no

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no

Classification of structural chromosomal aberrations included chromatid breaks and exchanges, chromosome breaks and exchanges (dicentric, cyclic chromasomes and such) and others (fragments and such). With numerical aberrations, only the polyploidal cells (polyploids) were recorded.
Gaps (chromosome gaps and chromatid gaps) were recorded as aberrations but were not included as structural aberrations. Gaps were those where the non stained portion of the chromatid was narrower than the width of the chromatid.
For the total number of chromosomal aberrations, cells with even a single aberration classified as stated above were recorded as cells with aberrations, and the total was tallied by classification of aberration.

Evaluation criteria:

See "statistics"

Statistics:

To determine the results of the study, the frequency of appearance of structural aberration and polyploidal cells was conducted using multiple sample x2 testing, and if a significant difference (5% or less of the significance level) was noted, the Fisher exact test method was used to determine the significance between the negative control group and each dose group (the significance level took into consideration the multiplicity, and separated the number of treatment groups into 5% and 1%). When the results were compared with the negative control group, the frequency of appearance of cells with chromosomal aberration in the test substance groups was significantly higher in two or more doses, and when the dose dependence was confirmed, it was determined that the chromosomal aberration inductivity was positive. When a significant increase was confirmed at a single dose, verification tests were conducted using a similar dose, and when the reproducibility results were confirmed, the chromosomal aberration inductivity was positive.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

1. Chromosomal Aberration Test/ Short Term Treatment Method: Without S9 Mix
The frequency of occurrence of cells with chromosome structural aberration was a low value of 2.0% in the negative control group. In the test substance groups, the range of frequency of occurrence was 0.5~4.0%, which was not statistically significant when compared to the negative control group. The frequency of occurrence of cells with chromosome structural aberration was 97.5% in the MNNG positive control group, and obvious chromosomal aberration induction was confirmed.
A low frequency of occurrence of 0.5% was confirmed for polyploids showing numerical aberration in the negative control group. A low frequency of occurrence in the range of 0~1.0% was confirmed for the test substance groups. Polyploids were not verified in the positive control group.
In the 600 µg/mL dose group, observable metaphase division was not noted due to the toxicity on the test substance cells.

2. Chromosomal Aberration Test/ Short Term Treatment Method: With S9 Mix
The frequency of occurrence of cells with chromosome structural aberration was a low value of 1.5% in the negative control group. In the test substance groups, the frequencies of occurrence for 75, 150, 300, 450 and 600 µg/mL were 0.5, 0, 0.5, 0.5 and 7.0%, respectively, and the frequency of occurrence for 600 µg/mL increased significantly when compared to the negative control group. The frequency of occurrence of cells with chromosome structural aberration was 55.0% in the B[a]P positive control group, and remarkable chromosomal aberration induction was confirmed.
Polyploids were not noted in the negative control group or the positive control group. A low frequency of occurrence in the range of 0~1.5% was confirmed for the test substance groups.

3. Chromosomal Aberration Test/ Short Term Treatment Method: With S9 Mix – Verification Test
Since there was a significant increase in cells with structural aberration in only one dose (600 µg/mL) of in the short term treatment method with the S9 mix, three doses of 400, 600 and 800 µg/mL were established and verification tests were conducted with the S9 mix using the short term treatment method. The frequency of occurrence of cells with chromosome structural aberration was a low value of 0.5% in the negative control group. In the test substance groups, the frequencies of occurrence for 400 and 600 µg/mL were 2.5 and 9.0%, respectively, and only the frequency of occurrence for 600 µg/mL increased significantly. The frequency of occurrence of cells with chromosome structural aberration was 52.0% in the B[a]P positive control group, and remarkable chromosomal aberration induction was verified.
A low frequency of occurrence of 0.5% was confirmed for polyploids in the negative control group. Low frequencies of occurrence of 1.0 and 2.5% were confirmed for the test substance groups. Polyploids were not verified in the positive control group.
In the 800 µg/mL dose group, observable metaphase division was not noted due to the toxicity on the cells.

Any other information on results incl. tables

Table no.1 : Results of chromosomal aberration tests on N,N’-bis(2-Methylphenyl) guanidine / Short term treatment method : WITHOUT S9 mix.

Dose (ug/ml)	Number of cells with structural chromosomal aberration (%)						Occurrence of gaps (%)	Cell growth rate (%)	Number of cells with numerical chromosomal aberration ( %)
	N	chromatid		chromosome		Total			N	Polyploid
		B	E	B	E
0	400	1	1	0	0	2%	0	100	400	0.5
75	400	0.5	0.5	0	0	0.5%	0	96.5	400	0
150	400	0	0	0	0.5	0.5%	0	97	400	0.5
300	400	0	0.5	0	0	0.5%	0	89	400	1
450	400	1.5	2.5	0	0	4%	0	69.5	400	0.5
600#	-	-	-	-	-	-	-	32	-	-
Positive control	400	42	95.9	1	0.5	97.5%**	1	-	400	0

N = Number of cells observed, B= break, E= exchange

# Due to the cytotoxicity, observable mid-split images were not evident.

** p<0.01

 

Table no.2 : Results of chromosomal aberration tests on N,N’-bis(2-Methylphenyl) guanidine / Short term treatment method : WITH S9 mix.

Dose (ug/ml)	Number of cells with structural chromosomal aberration (%)						Occurrence of gaps (%)	Cell growth rate (%)	Number of cells with numerical chromosomal aberration ( %)
	N	chromatid		chromosome		Total			N	Polyploid
		B	E	B	E
0	400	0.5	1.5	0	0	1.5%	0	100	400	0
75	400	0.5	0	0	0	0.5%	0.5	78	400	0
150	400	0	0	0	0	0%	0.5	69.5	400	0
300	400	0	0.5	0	0	0.5%	0	64.5	400	0.5
450	400	0	0.5	0	0	0.5%	0	44	400	1.5
600	400	3	6	0.5	0.5	7%*	0	44	400	1.5
Positive control	400	6.5	54	0	0.5	55%**	0	-	400	0

N = Number of cells observed

** p<0.01

 

Table no.3 : Results of chromosomal aberration tests on N,N’-bis(2-Methylphenyl) guanidine / Short term treatment method : WITH S9 mix (verification test)

Dose (ug/ml)	Number of cells with structural chromosomal aberration (%)						Occurrence of gaps (%)	Cell growth rate (%)	Number of cells with numerical chromosomal aberration ( %)
	N	chromatid		chromosome		Total			N	Polyploid
		B	E	B	E
0	400	0	0	0	0.5	0.5%	0	100	400	0.5
400	400	0	2	0	0.5	2.5%	0	69.5	400	1
600	400	5	7	0	1.0	9%**	0	34.5	400	2.5
800#	400	-	-	-	-	-	-	14.5	400	-
Positive control	400	11	48.5	0	0	52%**	0	-	400	0

N = Number of cells observed

** p<0.01

# Due to the cytotoxicity, observable mid-split images were not evident.

Applicant's summary and conclusion

Conclusions:

To study the presence of chromosomal aberration induction relating to N, N’-bis (2-methylphenyl) guanidine, in vitro chromosomal aberration tests were conducted using Chinese hamster lung fibroblasts (CHL/IU) during short term treatment tests. The results verified a significant increase in the number of cells with chromosome structural aberrations during short term treatment tests with the S9 mix, and also verified the reproducibility of the results.
Under the conditions of this study, we have determined positive chromosomal aberration induction of N, N’-bis (2-methylphenyl) guanidine on CHL/IU cells.

Executive summary:

To study the presence of chromosomal aberration induction relating to N, N’-bis (2-methylphenyl) guanidine,in vitrochromosomal aberration tests were conducted using Chinese hamster lung fibroblasts (CHL/IU) during short term treatment tests.

To set the doses used in the chromosomal aberration tests, cell growth inhibition tests were performed in a range of 37.5~2400µg/mL. This resulted in confirmation of cell growth inhibition of more than 50% at a dose of 600 µg/mL and higher with and without the S9 mix.

Therefore, the doses used for the chromosomal aberration tests were 75, 150, 300, 450 and 600 µg/mL.

The results of the study confirmed a significant increase in cells with structural chromosomal aberrations at 600 µg/mL with the S9 mix. A significant increase in cells with structural chromosomal aberrations was confirmed at 600 µg/mL even in verification tests with the S9 mix performed using doses of 400, 600 and 800 µg/mL, and at 800 µg/mL, observable mid-split images were not evident due to the cytotoxicity.

Based on the aforementioned results, it was determined that the chromosomal aberration induction was positive for N, N’-bis (2-methylphenyl) guanidine on CHL/IU cells under these test conditions. The D₂₀value of this test substance was 2.73 mg/mL.