[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA (OECD TG 471) (Genetic Laboratory, JBC Inc., 1994).

Cytogenicity in mammalian cells: positive with metabolic activation in Chinese hamster lung fibroblasts (similar to OECD 473) (Hita laboratories, 1996). Although induction of chromosomal aberration was not reported in the study without metabolic activation, structural aberrations at 500-800 µg/ml and numerical aberrations at 500 µg/ml in 18-h recovery the group were observed when tested in the presence of metabolic activation. Chromosomal aberrations were induced by the treatment with the positive control substances, mitomycin C and cyclophosphamide. No concurrent cytotoxicity data are presented in the report. The pre-experiment for toxicity showed a reduction of mitotic index to approximately 50% at 500 µg/ml. The aberrations observed at 800 µg/ml are very possibly the result of excessive toxicity, and it is possible that the 500 µg/ml results are also a result of toxicity. Therefore, it is not possible to fully evaluate the results.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 July - 19 August 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

other: Standards for Mutagenicity Test using Microorganisms (JMOL)

Qualifier:

according to guideline

Guideline:

other: Guidelines for Screening Toxicity Testing of Chemicals and Guidelines for Toxicity Testing of Chemicals (JMITI)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

tryptophan (E. coli strain) and histidine (Salmonella strains)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and 5,6-benzoflavone, S9 induced rat liver

Test concentrations with justification for top dose:

Preliminary test: 10, 50, 100, 500, 1000, 5000 µg/plate
Main test: 313, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test substance was found to be soluble in DMSO and acetone, but not in water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: AF-2; 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

-MA: TA 100, WP2 uvrA - 0.01 µg/plate; TA 98 - 0.1 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

-MA: TA 1535 - 0.5 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

-MA: TA 1537 - 80.0 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-AA (2-Aminoanthracene)

Remarks:

+MA: TA 1535 - 2.0 µg/plate; WP2 uvrA - 10.0 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

+MA: TA 100, TA 98, TA 1537 - 5.0 µg/plate

Details on test system and experimental conditions:

ACTIVATION:
S9 mix per ml:
S9 fraction: 10 v/v%
MgCl2: 8 µmol
KCl: 33 µmol
G-6-P: 5 µmol
NADPH: 4 µmol
NADH: 4 µmol
0.5 ml S9 mix was added to a total volume of 2.7 ml, giving a final concentration of approximately 2% S9.


METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 37°C for 48 hours

SELECTION AGENT (mutation assays): minimum glucose agar

NUMBER OF REPLICATIONS: triplicate plates, experiment repeated

DETERMINATION OF CYTOTOXICITY
- increase in number of revertant colonies

Evaluation criteria:

The number of revertant colonies of each test group were compared with and analysed for a statistically significant increase (p<0.01) from that of the negative control group at each dose in each bacterial strain with and without metabolic activation.

Statistics:

Dunnett's multiple comparison (one-sided test)
Linear regression

Key result

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Observations:
The test substance produced neither toxic effects nor a statistically significant increase in the number of revertant colonies in any conditions in any bacterial strains.

All of the positive controls produced significant increases in the number of revertant colonies in all bacterial strains with and without metabolic activation. This shows the test method employed in the test is effective for the purpose.

Remarks on result:

other: all strains/cell types tested

Table 1: Preliminary test - number of revertants per plate (mean of 2 plates)

Dose (µg/plate)	TA 100	TA 100	TA 1535	TA 1535	WP2 uvrA	WP2 uvrA	TA 98	TA 98	TA 1537	TA 1537
	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA
DMSO	133	124	9	11	28	31	26	29	10	10
10	133	115	9	13	24	30	26	27	8	12
50	126	120	11	11	24	29	26	34	8	9
100	123	116	8	14	30	35	20	30	7	12
500	135	133	9	14	23	32	21	36	9	9
1000	127	139	12	10	30	32	22	27	6	10
5000	126	138	14	10	29	36	27	36	5	5
Positive control	372	1227	301	413	99	733	497	277	730	53

 

Table 2: Main test 1 - number of revertants per plate (mean of 3 plates)

Dose (µg/plate)	TA 100	TA 100	TA 1535	TA 1535	WP2 uvrA	WP2 uvrA	TA 98	TA 98	TA 1537	TA 1537
	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA
DMSO	117	128	11	12	29	30	20	24	6	10
313	110	137	13	14	27	37	22	25	5	7
625	110	142	11	11	24	37	23	29	6	8
1250	111	128	9	12	29	31	21	32	8	8
2500	110	128	11	14	25	35	27	30	5	8
5000	116	116	10	12	27	37	23	25	5	6
Positive control	354	1178	285	444	85	818	457	273	990	55

 

Table 3: Main test 2 - number of revertants per plate (mean of 3 plates)

Dose (µg/plate)	TA 100	TA 100	TA 1535	TA 1535	WP2 uvrA	WP2 uvrA	TA 98	TA 98	TA 1537	TA 1537
	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA
DMSO	115	122	11	12	25	33	2.5	28	7	11
313	112	121	10	11	29	34	2.3	31	8	10
625	124	135	9	12	30	36	1.5	30	8	8
1250	107	136	9	14	22	34	3.0	29	6	8
2500	119	117	9	10	27	33	6.0	23	8	9
5000	113	122	11	9	28	32	1.5	26	9	7
Positive control	438	1334	223	437	111	1087	456	273	1077	56

 

Conclusions:

The test substance has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD TG 471 and 472 and in compliant with GLP. No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or E. coli WP2 uvrA in the initial or the repeat experiments up to limit concentrations. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The restrictions were that the number of cells evaluated does not comply with the current guideline. An audited translation from Japanese was available to the reviewer, date of translation November 12 1998.

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Annex V (In Vitro Cytogenetics)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

The number of cells evaluated does not comply with the current guideline. Concurrent cytogenicity data are not included in the study report.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: Chinese Hamster Lung fibroblasts (CHL cells; clone 11)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and 5,6-benzoflavone.

Test concentrations with justification for top dose:

Experiment 1 and 2: 25, 50, 100 µg/ml (without metabolic activation); 200, 500, 800 µg/ml (with metabolic activation)

Vehicle / solvent:

Dimethyl sulphoxide

Details on test system and experimental conditions:

ACTIVATION: S9 mix included magnesium chloride, potassium chloride, Glucose-6-phosphate and NADP

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 hours (with metabolic activation); 24 and 48 hours (without metabolic activation)

Fixation time:
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hours, with and without metabolic activation

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

NUMBER OF REPLICATIONS: duplicate cultures, experiment repeated

NUMBER OF CELLS EVALUATED: chromosome aberrations were evaluated in 200 metaphases per concentration; mitotic index was determined from 100 cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

Structural aberrations (not including gaps) compared with negative control and judged to be: negative if incidence not increased with a significance level of 5%; positive if incidence not increased with a significance level of 5% and results reproducible.

Statistics:

Fischer's exact probability test.

Key result

Species / strain:

mammalian cell line, other: Chinese Hamster Lung fibroblasts (CHL cells; clone 11)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

mitotic index reduced to approximately 50% of control at 500 µg/ml in the preliminary cytotoxicity study

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mammalian cell line, other: Chinese Hamster Lung fibroblasts (CHL cells; clone 11)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

mitotic index reduced to approximately 20% (18 h recovery) or 50% (48 hour recovery) of control at 100 µg/ml in the preliminary cytotoxicity study

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The preliminary cell growth and cell division inhibition test indicated that without metabolic activation 50% growth inhibition occurred at about 85 µg/ml (6 h exposure); at about 75 µg/ml (24 h exposure) and at about 65 µg/ml (48 h exposure); with metabolic activation 50% growth inhibition occurred at about 500 µg/ml.

Observations: The number of cells with numerical aberrations (polyploidy and endoreduplication) and chromatid type or chromosome type of structural aberration (gap ,break, exchange, etc.) was counted by observing 50 cells per slide and total 200 metaphases per each concentrations, and each incidence of the cells with the numerical and/or structural aberration was recorded. Total incidence of the cells with any aberration in each concentration was recorded in 2 ways of inclusion of gap or not. A gap was defined as a clear discontinuity, wider than one chromatid but narrower than two, accompanied with a minimal misalignment of the chromatid. Slides were all coded and blind-tested by microscopy. The mitotic rates for each group, except for the positive control, were calculated from the results of observation of 500 cells per dish, to make a total of 1000 cells per concentration.

Although induction of chromosomal aberration was not reported in the study without metabolic activation, structural aberrations at 500-800 µg/ml and numerical aberrations at 500 µg/ml in 18-h recovery the group were observed when tested in the presence of metabolic activation. Chromosomal aberrations were induced by the treatment with the positive control substances, mitomycin C and cyclophosphamide. No concurrent cytotoxicity data are presented in the report. The pre-experiment for toxicity showed a reduction of mitotic index to approximately 50% at 500 µg/ml. The aberrations observed at 800 µg/ml are very possibly the result of excessive toxicity, and it is possible that the 500 µg/ml results are also a result of toxicity. Therefore, it is not possible to fully evaluate the results.

Table 1: Results of the dose range finding test Direct method (without metabolic activation)

Concentration of test substance (µg/ml)	Cell growth rate	Cell growth rate	Mitotic rate	Mitotic rate
	24 h-treatment	48 h-treatment	24 h-treatment	48 h-treatment
0	100	100	100	100
20	94.9	102.8	92.7	80.0
40	89.0	120.4	122.0	91.4
60	86.9	105.1	129.3	57.1
80	39.4	31.2	65.9	54.3
100	22.0	16.8	22.0	51.4
200	14.5	14.4	9.8	34.3
300	16.1	2.5	-	11.4
500	17.7	3.3	-	-
1000	11.5	3.2	-	-

 

Table 2: Results of the dose range finding test (with and without metabolic activation

Concentration of test substance (µg/ml)	Cell growth rate	Cell growth rate	Cell growth rate	Mitotic rate	Mitotic rate	Mitotic rate
	-MA 18 h-recovery	+MA 18 h-recover	+MA 42 h-recovery	-MA 18 h-recovery	+MA 18 h-recover	+MA 42 h-recovery
0	100	100	100	100	100	100
40	94.0	-	-	92.6	-	-
60	93.6	-	-	81.5	-	-
80	60.0	-	-	74.1	-	-
100	22.1	90.7	96.8	44.1	90.7	80.9
200	6.4	89.5	93.5	-	89.5	104.3
300	3.0	82.9	78.0	-	82.9	89.4
500	3.4	54.2	51.1	-	54.2	87.2
800	1.3	10.5	20.9	-	10.5	48.9
1000*	2.1	0.9	0.6	-	-	-
1500*	-	0.9	0.3	-	-	-
2000*	-	0.6	0.3	-	-	-
3000*	-	0.9	0.4	-	-	-

*precipitation of the test substance was observed

 

Table 3: Results of chromosome aberration test (first trial, without metabolic activation)

Treatment time (h)	Concentration (µg/ml)	Number of polyploid cells	Percentage cells with aberrations	Percentage cells with aberrations
			-g	+g
24	Solvent control	0	0	0
48	Solvent control	0	1	1
24	25 50 100	2 2 3	5* 2 1	5* 2 1
48	25 50 100	0 1 6*	2 0 2	2 1 2
24	Positive control	0	113	114
48	Positive control	1	129	130

 * significant at p < 0.05

 

Table 4: Results of chromosome aberration test (first trial, with and without metabolic activation)

S9 mix	Concentration (µg/ml)	Number of polyploid cells	Percentage cells with aberrations	Percentage cells with aberrations
			-g	+g
-MA	Solvent control	0	1	1
+MA 18 h-recovery	Solvent control	0	1	1
-MA	25 50 100	0 0 0	2 1 2	2 2 2
+MA 18 h-recovery	200 500 800	1 16** 5*	0 41** 51**	0 42** 54**
-	Positive control	1	104	105
+MA 18 h-recovery	Positive control	2	70	72

 * significant at p < 0.05

* * significant at p < 0.01

 Table 5: Results of chromosome aberration test (first trial, with metabolic activation)

S9 mix	Concentration (µg/ml)	Number of polyploid cells	Percentage cells with aberrations	Percentage cells with aberrations
			-g	+g
+MA 42 h-recovery	Solvent control	0	1	1
+MA 42 h-recovery	200 500 800	3 3 3	0 2 4	0 2 4
+ 42 h-recovery	Positive control	1	8	8

Table 6: Results of chromosome aberration test (second trial, without metabolic activation)

Treatment time (h)	Concentration (µg/ml)	Number of polyploid cells	Percentage cells with aberrations	Percentage cells with aberrations
			-g	+g
24	Solvent control	0	2	3
48	Solvent control	3	2	2
24	25 50 100	2 0 0	2 1 3	2 1 3
48	25 50 100	1 0 0	1 0 0	1 0 0
24	Positive control	0	105	105
48	Positive control	0	85	87

 

Table 7: Results of chromosome aberration test (second trial, with and without metabolic activation)

S9 mix	Concentration (µg/ml)	Number of polyploid cells	Percentage cells with aberrations	Percentage cells with aberrations
			-g	+g
-MA	Solvent control	1	1	1
+MA 18 h-recovery	Solvent control	0	0	0
-MA	25 50 100	1 1 1	3 1 0	4 1 0
+MA 18 h-recovery	200 500 800	1 26** 17**	10 54** 55**	11 57** 56**
-MA	Positive control	1	90	90
+MA 18 h-recovery	Positive control	0	88	89

 ** significant at p < 0.01

 

Table 8: Results of chromosome aberration test (second trial, with metabolic activation)

S9 mix	Concentration (µg/ml)	Number of polyploid cells	Percentage of cells with aberrations	Percentage of cells with aberrations
			-g	+g
+MA 42 h-recovery	Solvent control	4	2	2
+MA 42 h-recovery	200 500 800	0 4 13*	4 6 3	4 6 3
+MA 42 h-recovery	Positive control	3	10	10

 * significant at p < 0.05

Conclusions:

The substance has been tested for cytogenicity in mammalian cells in a study conducted according to a Japanese guideline similar to OECD 473 and in compliance with GLP. A statistically significant dose-dependent increase in the number of cells with structural aberrations and with polyploidy was observed in the presence of metabolic activation with 18 hours recovery in the initial and the repeat experiment. In cultures treated in the presence of metabolic activation with a 42 hours recovery period, no increase in structural aberrations was observed, though a statistically significant increase in polyploidy was reported at the highest dose tested. Only preliminary cytotoxicity data are available, so it is not possible to evaluate whether the increase in numerical aberration was a result of toxicity. No increase in numerical or structural aberrations was reported in the absence of metabolic activation. It is concluded that the test substance may be positive for cytogenicity under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus assay in ICR mouse (oral administration): negative (OECD 474) (Korea Institute of Toxicology, 2004).

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:

29 June 2004 to 16 July 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Orient C., Ltd., Seoul, Korea
- Age at study initiation: six weeks
- Weight at study initiation: 23.5-27.0 g
- Assigned to test groups randomly: using algorithm of Path/Tox so that each group had similar body weight distribution
- Fasting period before study:
- Housing: up to six animals per cage in polycarbonate cages with bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23°C ± 3°C
- Humidity (%): 50%
- Air changes (per hr): 10-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: mid-May 2004 To: July 8 2004

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: test item is insoluble in water
- Concentration of test material in vehicle: no information
- Amount of vehicle (if gavage or dermal): no information. It is not stated if administration was by gavage, but this is presumed to be the case

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Two treatments at 24 hour intervals

Remarks:

Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:

No. of animals per sex per dose:

Six males

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control substance: cyclophosphamide
- Justification for choice of positive control(s): widely used in micronucleus assays
- Route of administration: intraperitoneal
- Doses / concentrations: 70 mg/kg bw

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: preliminary toxicity study

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): animals were sacrificed 24 hours after the second administration

DETAILS OF SLIDE PREPARATION: cells were collected in fetal bovine serum, centrifuged and smeared onto slides. Preparations were dried and fixed by submerging in methanol. Slides were stained with May-Grunwald and Giesma stains, then rinsed dried and mounted.

METHOD OF ANALYSIS: Slides were examined under x1000 magnification. Small round or oval bodies (1/5 to 1/20 diameter of PCE) were counted as micronuclei. Micronuclei were counted in 200 PCE's. The (PCE):(PCE+NCE) ratio was calculated by counting 500 cells

Evaluation criteria:

The result was judged as positive if there was a statistically significant and dose-related increase or a reproducible increase in the frequency of MNPCEs at least at one dose level.

Statistics:

Differences of numbers of MNPCRs between treated and vehicle control: Kruskal-Wallis' H-test and Dunnett's test.
Differences of numbers of MNPCRs between positive and vehicle control: Mann-Whitney's U-test.
Differences of PCE/(PCE+NCE) between treated and vehicle control:

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

No effects observed in bone marrow, and no clinical signs were noted

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Table 1: Micronucleus test in male ICR mice

Chemical treated	Dose (mg/kg)	No. of animals	MNPCE/2000 PCEs	PCE/(PCE+NCE)
Vehicle	0	6	1.17	0.52
Test item	500	6	1.33	0.49
Test item	1000	6	1.83	0.55
Test item	2000	6	2.00	0.55
CPA	70	6	94.83**	0.45*

* Significantly different from the control at P <0.01

** Significantly different from the control at P < 0.05

Conclusions:

The test substance has been tested in an in vivo micronucleus assay conducted according to OECD 474 and in compliance with GLP. No evidence of induction of micronuclei was observed in male ICR mice. No signs of systemic toxicity were observed and the test item did not cause toxicity in the bone marrow. Solvent and positive controls gave expected responses. It is concluded that the test substance is negative for the induction of micronuclei under the conditions of the study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The submission substance has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD TG 471 and OECD TG 472 and in compliance with GLP (Genetic Laboratory, JBC Inc., 1994). No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or E. coli WP2 uvrA in the initial or the repeat experiments up to limit concentrations. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

The submission substance has been tested for cytogenicity in mammalian cells in a study conducted according to a Japanese guideline similar to OECD TG 473 and in compliance with GLP using Chinese hamster lung fibroblasts (Hita laboratories, 1996). A statistically significant dose-dependent increase in the number of cells with structural aberrations and with polyploidy was observed in the presence of metabolic activation with 18 hours recovery in the initial and the repeat experiment. In cultures treated in the presence of metabolic activation with a 42 hours recovery period, no increase in structural aberrations was observed, though a statistically significant increase in polyploidy was reported but not at the highest dose tested. Only preliminary cytotoxicity data are available and not concurrent data. The pre-experiment for toxicity showed toxicity at 500 µg/ml (reduction of mitotic index to approximately 50%). The aberrations observed at 800 µg/ml are very possibly the result of excessive toxicity, and it is possible that the 500 µg/ml results are also a result of toxicity. Therefore, it is not possible to fully evaluate whether the increase in numerical aberration was a result of toxicity. No increase in numerical or structural aberrations was reported in the absence of metabolic activation. It is concluded that the test substance may be positive for cytogenicity under the conditions of the test.

The test substance has been tested in an in vivo micronucleus assay conducted according to OECD TG 474 and in compliance with GLP. No evidence of induction of micronuclei was observed in male ICR mice (Korea Institute of Toxicology, 2004). No signs of systemic toxicity were observed and the test item did not cause toxicity in the bone marrow. Solvent and positive controls gave expected responses. It is concluded that the test substance is negative for the induction of micronuclei under the conditions of the study. There are no toxicokinetic studies for the test substance to confirm exposure of the bone marrow. However, the available repeated dose toxicity study revealed non-adverse signs of systemic availability, such as hepatic hypertrophy, changes to clinical chemistry parameters and kidney weight changes.

Justification for classification or non-classification

Based on the available data, classification for genetic toxicity is not required for the submission substance according to Regulation (EC) No 1272/2008.