1,3-di-o-tolylguanidine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test (OECD 471) and a mouse lymphoma assay (OECD 476) are available on DOTG and show negative result.
However, the chromosomal aberration test (OECD 473) on DOTG is positive with metabolic activation only.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

not specified

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

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.

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.

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

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.

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine for S. typhimurium strains, and tryptophan for E. coli strains

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

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 ; 0, 78.1, 156, 313, 625, 1250, 2500 µg/plate (TA100, TA1537)
Without S9 mix; 0, 156, 313, 625, 1250, 2500, 5000 µg/plate (TA98, TA1535, WP2 uvrA)
With S9 mix; 0, 156, 313, 625, 1250, 2500, 5000 µg/plate (all strains)

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.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

-S9 mix; 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (TA100, TA98, WP2 uvrA), Sodium azide (TA1535) and 9-Amino-acridine (TA1537) , +S9 mix; 2-Aminoanthracene (all strains)

Positive control substance:

other:

Details on test system and experimental conditions:

Plates/test : 3
Number of replicates : 2

(1) Pre-incubation Method (without metabolic activation)
0.1ml of the test substance solution, 0.5ml of a 0.1M sodium-phosphate buffer (pH of 7.4) and 0.1 mL of the pre-incubated bacteria suspension were combined and subject to repetitive shaking incubation for 20 minutes at 37°C.
2ml of the soft agar with amino acid maintained at 45°C was added and spread on minimal glucose agar plate media. The minimal glucose agar plate media (plate) involved 1.5 w/v% of powdered agar and 2 w/v% of glucose on Vogel-Bonner E medium, added in 30 mL quantities. This was incubated for 48 hours at 37°C and then the number of revertant colonies was calculated, and simultaneous observations were made on the presence of growth inhibition of the indicator strains using a microscope.

(2) Pre-incubation Method (with metabolic activation)
0.1ml of the test substance solution, 0.5ml of the S9 mix and 0.1 mL of the pre-incubated bacteria suspension were combined and subject to repetitive shaking incubation for 20 minutes at 37°C. 2ml of the soft agar with amino acid maintained at 45°C was added and spread on minimal glucose agar plate media. After incubating for 48 hours at 37°C, the number of revertant colonies was calculated, and simultaneous observations were made on the presence of growth inhibition of the indicator strains using a microscope.

The negative and positive controls were conducted in the same manner using 0.1 mL of the solvent (DMSO) and the positive control substance solution instead of 0.1 mL of the test substance solution.

Evaluation criteria:

If the following three standards were satisfied, the test was conducted under appropriate conditions and the test was deemed to be valid.
(1) There was no mixing of the various bacteria in the bacterial solutions, solvent, test substance solution or S9 mix used in these tests.
(2) The number of revertant colonies in the negative control for each of the indicator strains was a value within the range of the background data for this laboratory (number of natural mutations).
(3) The number of revertant colonies in the positive control for each of the indicator strains was a value close to or within the range of the background data for the positive control values for this laboratory.

Statistics:

no

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was observed at above 1250 µg/plate (TA1537), above 2500 µg/plate (TA98, TA1535) and 2500 µg/plate (TA100) without S9 mix, and above 2500 µg/plate (all strains) with S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was observed at above 2500 µg/plate without S9 mix, and above 2500 µg/plate (all strains) with S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The number of revertant colonies for all of the bacteria tested during testing performed two times as well as during both the direct method and the metabolic activation method was not more than double that of the negative control values.
Without metabolic activation, the doses of 1250 µg/plate and greater for TA1537, and 2500 µg/plate and greater for TA100 and doses of 2500 µg/plate and greater for TA98, TA1535 and WP2uvrA, and for the metabolic activation method, the doses of 2500 µg/plate for all of the test strains did not result in inhibition of the growth of the bacteria.
For the negative control group, the number of revertant colonies was confirmed to be within the range of background data.
For the positive control group, there was a clear increase in the number of revertant colonies, and those levels showed positive values within the ranges of the background data.
Furthermore, there was no verification of mixing of the various bacteria in the bacterial solutions, solvent, test substance solution or S9 mix used in these tests.
For both the direct method and the metabolic activation method, a white deposit was noted in the test tubes at the end of the pre-incubation period for the 5000 µg/plate but after layering on the plate, the deposit was not verified on the plate at the end of the specified incubation period.

Table 1: Results table / Ames test - First experiment without S9 (direct method)

 

strains	Doses (µg/plate)	Revertants per plate			mean	SD
TA 100	0	105	109	117	110	6
	78.1	103	94	90	96	7
	156	98	96	94	96	2
	313	108	105	86	100	12
	625	103	103	83	96	12
	1250	101	93	99	98	4
	2500	55*	60*	95*	70	22
	5000	-	-	-	-	-
	AF-2 (0.01)	730	701	717	716	15
TA 1535	0	10	13	7	10	3
	78.1	-	-	-	-	-
	156	10	9	10	10	1
	313	8	11	12	10	2
	625	5	14	12	10	5
	1250	15	7	13	12	4
	2500	4*	10*	3*	6	4
	5000	4*	0*	0*	1	2
	SA (0.5)	336	324	326	329	6
WP2uvrA	0	25	23	20	23	3
	78.1	-	-	-	-	-
	156	21	21	14	19	4
	313	16	21	22	20	3
	625	18	25	19	21	4
	1250	14	21	17	17	4
	2500	15*	17*	26*	19	6
	5000	9*	11*	9*	10	1
	AF-2 (0.04)	785	855	811	817	35
TA 98	0	15	17	27	20	6
	78.1	-	-	-	-	-
	156	19	23	24	22	3
	313	15	16	19	17	2
	625	13	16	20	16	4
	1250	21	18	15	18	3
	2500	14*	20*	6*	13	7
	5000	2*	0*	0*	1	1
	AF-2 (0.1)	403	471	475	450	40
TA 1537	0	12	6	8	9	3
	78.1	5	6	13	8	4
	156	11	6	11	9	3
	313	6	9	9	8	2
	625	10	13	7	10	3
	1250	13*	8*	7*	9	3
	2500	3*	0*	6*	3	3
	5000	-	-	-	-	-
	9-AA (80)	421	408	412	411	2

 

0: vehicle control (DMSO)

*: bacterial growth inhibition confirmed

SD: standard deviation

 

 

Table 2: Results table / Ames test - Second experiment without S9 (direct method)

 

strains	Doses (µg/plate)	Revertants per plate			mean	SD
TA 100	0	102	104	96	101	4
	78.1	102	95	116	104	11
	156	92	98	116	102	12
	313	105	115	98	106	9
	625	115	89	104	103	13
	1250	85	104	112	100	14
	2500	71*	66*	62*	66	5
	5000	-	-	-	-	-
	AF-2 (0.01)	676	663	705	681	22
TA 1535	0	5	10	6	7	3
	78.1	-	-	-	-	-
	156	14	8	7	10	4
	313	13	4	9	9	5
	625	7	8	16	10	5
	1250	9	7	9	8	1
	2500	10*	6*	4*	7	3
	5000	3*	3*	5*	4	1
	SA (0.5)	373	399	400	391	15
WP2uvrA	0	17	17	17	17	0
	78.1	-	-	-	-	-
	156	11	21	16	16	5
	313	27	22	22	24	3
	625	20	14	19	18	3
	1250	14	11	15	13	2
	2500	17*	10*	15*	14	4
	5000	16*	11*	8*	12	4
	AF-2 (0.04)	822	836	816	825	10
TA 98	0	18	14	20	17	3
	78.1	-	-	-	-	-
	156	9	14	20	14	6
	313	19	21	13	18	4
	625	15	15	22	17	4
	1250	8	27	17	17	10
	2500	17*	17*	12*	15	3
	5000	3*	5*	3*	4	1
	AF-2 (0.1)	532	509	503	515	15
TA 1537	0	7	5	5	6	1
	78.1	8	6	6	7	1
	156	5	7	4	5	2
	313	6	10	8	8	2
	625	4	6	6	5	1
	1250	4*	4*	4*	4	0
	2500	9*	4*	6*	6	3
	5000	-	-	-	-	-
	9-AA (80)	605	586	751	647	90

 

0: vehicle control (DMSO)

*: bacterial growth inhibition confirmed

SD: standard deviation

 

 

Conclusions:

From these aforementioned results, it has been determined that. N, N’-bis (2-methylphenyl) guanidine was negative for genetic mutation induction on bacteria.

Executive summary:

To study the presence of genetic mutagenicity of N, N’-bis (2-methylphenyl) guanidine, reverse mutation tests were performed using Salmonella typhimurium TA100, TA1535, TA98 and TA1537 as well as Escherichia coli WP2 uvrA and the indicator strains, performed with the pre-incubation method with (metabolic activation method) and without (direct method) the S9 mix.

Doses were based on the results of the dose setting tests (preliminary tests), and the dose confirmed to inhibit growth of the bacteria was deemed the maximum dose. For the direct method, the range was set to 78.1~2500µg/plate for TA100 and TA1537, and 156~5000µg/plate for TA98, TA1535 and WP2uvrA (ratio of 2). For the metabolic activation method, the range was set to 156~5000µg/plate for all the test strains (ratio of 2).

The study was performed twice. Based on the results, an increase in the number of reverse mutagenic colonies was not noted for any of the assay strains, regardless of the presence of metabolic activation. Growth inhibition of the strains was noted at doses of 1250µg/plate or greater of TA1537, 2500µg/plate or greater of TA 100 as well as with 2500µg/plate or greater of TA98, TA1535 and WP2uvrA when using the direct method, as well as with all of the strains at doses of 2500µg/plate or greater when using the metabolic activation method.

From these aforementioned results, it has been determined that. N, N’-bis (2-methylphenyl) guanidine was negative for genetic mutation induction on bacteria.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24/03/2011 – 18/07/2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK locus (trifluorothymidine resistance)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y mouse lymphoma cells, heterozygous (TK+/-) for the thymidine kinase enzyme system.
L5178Y TK+/- mouse lymphoma cells were obtained from ATCC (American Type Culture Collection - Rockville, MD 20852 - USA).
Periodically checked for Mycoplasma contamination: yes.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rat induced by Aroclor 1254

Test concentrations with justification for top dose:

Concentrations tested expressed as µg/mL pure 1,3-di-o-tolylguanidine
• Without S9 mix : 145.8 – 121.5 – 101.3 – 84.4 – 70.3 (assay 1, 3-hour treatment)
111.1 – 88.8 – 59.3 – 39.5 – 26.3 – 17.5 (assay 2, 24-hour treatment)
39.5 – 26.3 – 17.6 – 11.7 – 7.8 – 5.2 (assay 3, 24-hour treatment)

• With S9 mix :300 – 250 – 200 – 166.6 – 133.3 – 111.1 – 88.8 (assay 1)
250 – 200 – 166.6 – 133.3 – 111.1 (assay 2)

Vehicle / solvent:

As it was not soluble in aqueous solvent, the test item 1,3-di-o-tolylguanidine was dissolved in dimethylsulfoxyde (DMSO; Merck, batch K40013778 033). The stability of the test item in the solvent was unknown but preparations for treatment were performed just before use.
Successive dilutions were also performed in DMSO and used at 0.5 %.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methyl methanesulfonate (without S9), cyclophosphamide (with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium RPMI 1640

DURATION
- Pre-incubation:
Before each assay, a sufficient number of cells were thawed from the cryogenic ampoules, and cultured for 2 to 4 days in RPMI 10 medium in order to maintain exponential growth. The cells were then subcultured in 75 cm2 flasks at a determined cell density and incubated for 3 days at 37°C ± 0.5, humidity near 95% and 5% of CO2.
- Exposure duration: 3h or 24h without S9, and 3h with S9
- Expression time (cells in growth medium): 10-14 days

NUMBER OF REPLICATIONS: 2 per concentration

NUMBER OF CELLS EVALUATED:
After the 3-hour or 24-hour treatment, the surviving cells were counted by means of a hemocytometer. Part of the surviving cells were then re-plated in RPMI 20 medium (mean of 1.6 cells per well, 2 x 96- well microtiter plates per dose) and incubated at 37°C, humidity near 95% and 5% CO2.

DETERMINATION OF CYTOTOXICITY: YES, efficiency; relative total growth.
the remaining cells were incubated at a density of 2.105 cells/mL in RPMI 10 medium for 48 hours. After 24-hour incubation (day 1), cell counting was performed and if necessary, the cell density was adjusted at 2.105 cells/mL. Cell counting was repeated 24 hours later (day 2). The purpose of
these cell countings was to detect a possible toxicity of the test item on cell growth (Turner et al., 1984). New plating efficiencies (2 x 96-well microtiter plates per dose at 1.6 cells per well) were concurrently performed in order to calculate the relative total growth (RTG).

Evaluation criteria:

A study is accepted if:
-The plating efficiency of the negative control (PE2 %, mean of the 2 cultures), ranges from 65 to 120 % at T2 (Moore et al., 2006),
-The mutation frequency (MF) of the negative control is within the range of historical data of the laboratory, and ranges from 50 to 170 x10-6 mutants (Moore et al., 2006),
-The value of the suspension growth (SG) during the 2 day expression period of the negative control (i.e. SG = daily growth day 1 x daily growth day 2), ranges from 8 to 32 in the 3-hour treatment (Moore et al., 2006), and from 32 to 180 in the 24-hour treatment (San Francisco, EMS 6th harmonization meeting, 2005)-The induced mutation frequency (IMF) for the positive control is significantly increased when compared to the MF for the solvent control, and demonstrates an increase above the spontaneous background MF of at least 300 x10-6 mutants. Moreover, at least 40% of the IMF should be reflected in the small colonies (i.e. at least 120 x10-6 mutants; Moore et al., 2006). The observed values must be close to those of historical positive controls of the laboratory.

Statistics:

Statistical evaluation of data for the total number of mutants and for small colony mutants is performed using the method proposed by Robinson et al. (1990).

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY ASSAYs
In the preliminary cytotoxicity assay, the maximum dose was chosen function of the solubility of the test item, i.e. 750 µg/mL in final concentration.
Without metabolic activation after a 3-hour treatment , 1,3-di-o-tolylguanidine revealed a very strong cytotoxicity in L5178Y cells at the 3 highest concentrations tested from 750 to 187.5 µg/mL with no cell growth or very low adjusted RTG. The immediately inferior concentration of 93.75 µg/mL, induced a strong but acceptable level of cytotoxicity with an adjusted RTG of 18.9%. The concentration of 145.8 µg/mL was thus retained as the maximum concentration for the first mutagenicity test without S9-mix.
In the corresponding first mutagenicity assay, this concentration induced a moderate toxicity with an adjusted RTG of 32.8%, and was thus assessed for mutagenicity.

With metabolic activation, a very strong cytotoxicity in L5178Y cells at the 3 highest concentrations tested from 750 to 187.5 µg/mL was noted with no cell growth or very low adjusted RTG. The immediately inferior concentration of 93.75 µg/mL, induced a moderate level of cytotoxicity with an adjusted RTG of 63.7%. The concentration of 175 µg/mL was thus retained as the maximum concentration for the first mutagenicity test with S9-mix. However, this assay had to be reiterated. Indeed, the mutation frequency for the negative control was very higher than the maximal value already observed in historical data for negative control. Otherwise, during this assay, no toxicity anymore was noted at the highest dose tested of 175 µg/mL, and the reiterated assay was performed with an increased top concentration, i.e. 300 µg/mL. In the corresponding mutagenicity assay, the concentrations ranging from 111.1 to 59.3 µg/mL hindered cell growth. The immediately lower concentration of 39.5 µg/mL induced a strong toxicity with an adjusted RTG of 3.6 %, and the concentration of 26.3 µg/mL induced an adjusted RTG comprised between 10 and 20% with a value of 16.6%. At final, in the reiterated mutagenicity assay, the concentration of 39.5 µg/mL induced a strong toxicity with an adjusted RTG of 4.3 %, and the concentration of 26.3 µg/mL induced an adjusted RTG of 25.6%.

ACCEPTANCE CRITERIA FOR THE RESULTS
Concurrently to the main assays, tests were carried out with reference mutagenic compounds (methyl methanesulfonate in the absence of metabolic activation and cyclophosphamide in the presence of metabolic activation via S9-mix).
The plating efficiency of the negative control (mean of the 2 cultures) ranged from 65 to 120 % at T2 (Tables 14, 16, 20 and 22).
The mutation frequency (MF) of the negative control was within the range of historical data of the laboratory, and ranged from 50 to 170 x10-6 mutants
The suspension growth value of the negative control ranged from 8 to 32 in the 3-hour treatments, and were above 32 in the 24-hour treatment.
The induced mutation frequencies (IMF) for the positive controls were significantly increased when compared to the MF for the solvent control, and demonstrated an increase above the spontaneous background MF of at least 300 x10-6 mutants. Moreover, at least 40% of the IMF were reflected in the small colonies (i.e. at least 120 x10-6 mutants). The observed values were within or close to the limits
of historical positive controls of the laboratory, except in the 1st assay in absence of S9, in which the value was of 890.8 x10-6 mutants vs. 875.2 x10-6 mutants for the highest value already observed in
historic data. It was however considered that this deviation did not impair the current study. The acceptance criteria for the results were thus fulfilled.

Table of results : Mouse lymphoma test

 

ASSAY 1 WITHOUT S9 (3-hour treatment)	Conc (µg/mL)	0	70.3	84.4	101.3	121.5	145.8	MMS10
	Adjusted RTG	100	63.0	74.8	65.5	42.9	32.8	57.2
	IMF	-	31.1	4.6	11.9	21.7	38.6	800.7
	Statistical significance (Dunnett’s test)	-	N.S.	N.S.	N.S.	N.S.	N.S.	< 0.05

 

ASSAY 2 WITHOUT S9 (24-hour treatment)	Conc (µg/mL)	0	17.5	26.3	39.5	MMS 2
	Adjusted RTG	100	36.5	16.6	3.6	105.4
	IMF	-	-3.9	-17.5	22.6	354.9
	Statistical significance (Dunnett’s test)	-	N.S.	N.S.	*	<0.05

* Because of heterogeneity between two cultures, statistical analysis could not be assessed.

 

ASSAY 3 WITHOUT S9 (24-hour treatment)	Conc (µg/mL)	0	5.2	7.8	11.7	17.6	26.3	39.5	MMS 2
	Adjusted RTG	100	76.9	85.1	68.3	60.6	25.6	4.3	81.8
	IMF	-	-2.7	-0.7	21.2	-2.6	15.9	19.6	435.9
	Statistical significance (Dunnett’s test)	-	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	< 0.05

 

 

ASSAY 1 WITH S9 (3-hour treatment)	Conc (µg/mL)	0	88.8	111.1	133.3	166.6	200	CPA 2
	Adjusted RTG	100	92.1	63.5	67.1	32.1	15.9	73.3
	IMF	-	-8.6	6.4	-19.4	4.9	19.7	624.2
	Statistical significance (Dunnett’s test)	-	N.S.	N.S.	N.S.	N.S.	N.S.	< 0.05

 

 

ASSAY 2 WITH S9 (3-hour treatment)	Conc (µg/mL)	0	111.1	333.3	166.6	200	250	CPA 2
	Adjusted RTG	100	55.4	45.3	33.6	12.6	2.2	67.9
	IMF	-	-9.6	-2.8	18.7	42.3	**	510.6
	Statistical significance (Dunnett’s test)	-	N.S.	N.S.	*	N.S.	**	< 0.05

 

* Because of heterogeneity between two cultures, statistical analysis could not be assessed.

** Adjusted RTG being inferior to the limits accepted for mutagenicity analysis, this concentration was not taken into account in the interpretation of the resuts

Conclusions:

Under these experimental conditions, the test item 1,3-di-o-tolylguanidine induced no mutagenic activity at the TK locus in L5178Y mouse lymphoma cell culture either with or without metabolic activation, in two independent assays.

Executive summary:

The search for any mutagenic activity of 1,3-di-o-tolylguanidine was studied by means of gene mutation test at the TK locus in L5178Y mouse lymphoma cell culture in 2 and 3 independent assays with and without metabolic activation, respectively. DMSO was used as solvent. Cytotoxicity was observed in the main study after 24 -h treatment without S9 and after 3 -h treatment with S9.

In absence or in presence of metabolic activation neither statistically nor biologically significant increase in the mutation frequency of total mutants was noted at any of the concentration assessed of 1,3-di-o- tolylguanidine. Furthermore, no statistically significant increase in the mean number of small colonies and in the mutation frequency of small colony mutants was noted.

The acceptance criteria for the assay were fulfilled. The current study was considered as valid.

Under these experimental conditions, the test item 1,3-di-o-tolylguanidine induced no mutagenic activity at the TK locus in L5178Y mouse lymphoma cell culture either with or without metabolic activation, in two independent assays.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

1,3-di-o-tolyguanidine was judged not to be clastogenic in vivo in the micronucleus test performed in mice (OECD 474).

Link to relevant study records

Reference

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

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

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

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

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%.

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.

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.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test on DOTG (2002) - OECD 471 :

Reverse mutation tests were performed on DOTG using Salmonella typhimurium TA100, TA1535, TA98 and TA1537 as well as Escherichia coli WP2 uvrA and the indicator strains, performed with the pre-incubation method with (metabolic activation method) and without (direct method) the S9 mix.

The study was performed twice. Based on the results, an increase in the number of reverse mutagenic colonies was not noted for any of the assay strains, regardless of the presence of metabolic activation. Growth inhibition of the strains was noted at doses of 1250 µg/plate or greater of TA1537, 2500 µg/plate or greater of TA 100 as well as with 2500 µg/plate or greater of TA98, TA1535 and WP2uvrA when using the direct method, as well as with all of the strains at doses of 2500µg/plate or greater when using the metabolic activation method. From these aforementioned results, it has been determined that DOTG was negative for genetic mutation induction on bacteria.

 

Chromosomal aberration in vitro test on DOTG (2002) - OECD 473 :

In vitro chromosomal aberration tests were conducted on DOTG using Chinese hamster lung fibroblasts (CHL/IU) during short term treatment tests. 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 DOTG on CHL/IU cells under these test conditions.

 

Mouse lymphoma assay : in vitro mammalian cell gene mutation test on DOTG (2011) - OECD 476 :

The search for any mutagenic activity of 1,3-di-o-tolylguanidine was studied by means of gene mutation test at the TK locus in L5178Y mouse lymphoma cell culture in 2 and 3 independent assays with and without metabolic activation, respectively. DMSO was used as solvent. Cytotoxicity was observed in the main study after 24 -h treatment without S9 and after 3 -h treatment with S9.

In absence or in presence of metabolic activation neither statistically nor biologically significant increase in the mutation frequency of total mutants was noted at any of the concentration assessed of 1,3-di-o- tolylguanidine. Furthermore, no statistically significant increase in the mean number of small colonies and in the mutation frequency of small colony mutants was noted.

The acceptance criteria for the assay were fulfilled. The current study was considered as valid.

Under these experimental conditions, the test item 1,3-di-o-tolylguanidine induced no mutagenic activity at the TK locus in L5178Y mouse lymphoma cell culture either with or without metabolic activation, in two independent assays.

In vivo micronucleus study on DOTG - OECD 474 :

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.

 

Justification for classification or non-classification

 Based on the available data, 1,3-di-o-tolylguanidine should not be classified for genetic toxicity according to the Regulation EC N°1272/2008.