2,2-bis[(allyloxy)methyl]butyl (3-{[({2,2-bis[(allyloxy)methyl]butoxy}carbonyl)amino]methyl}-3,5,5-trimethylcyclohexyl)carbamate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Chromosome aberrations in CHL

Under the conditions of the study, the test material was concluded to be negative for the induction of structural or numerical chromosome aberrations in both non-activated and S9 activated test systems in the in vitro mammalian chromosome aberration test using CHL cells. The test material was not considered to be clastogenic in this in vitro study.

Ames test

Under the conditions of the study the test material was not mutagenic with Salmonella typhimurium test strains TA97, TA98, TA100 and TA102 in either the presence or absence of S9 metabolic activation.

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

Study period:

11 August 2009 to 21 September 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidelines for the testing of chemicals Section 4: Health Effects, Ministry of environmental protection of People's Republic of China, 473.

Version / remarks:

2003

Deviations:

no

GLP compliance:

not specified

Remarks:

Not specified

Type of assay:

other: chromosome aberration study in mammalian cells

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Preliminary study: 9.75, 19.5, 39.0, 78.1, 156.2, 312.5, 625, 1250, 2500 and 5000 µg/mL
Main study: without S9: 80, 160 and 320 µg/mL; with S9: 60, 120 and 240 µg/mL.

The test material dose levels selected for testing in the chromosome aberration assay were as a result of the preliminary toxicity assay.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

ACTIVATION SYSTEM
- The S9 was patch prepared by the supplier and stored at - 70 °C until use in this facility.
- Immediately prior to use, the S9 was thawed and mixed with a cofactor pool to contain as final concentrations in the S9 activated treatment medium with 8 mM MgCl2, 33 mM KCI, 5 mM glucose-6-phosphate, 4 mM nicotinamide-adenine dinucleotide phosphate and 100 µL S9 per millilitre medium.

PRELIMINARY TOXICITY ASSAY
- The concentration range covered 9.75 to 5000 µg/mL in the preliminary toxicity assay.

CHROMOSOME ABERRATION ASSAY
- The medium pH of this concentration was approximately 7.0.
- Cells were treated for 3 and 24 hours without metabolic activation (-S9- mix) and for 3 hours only with metabolic activation (+ S9- mix).
- 200 cells were counted per solvent control group
- 100 cells were counted per positive control group
- 200 cells were counted per test material treatment group

DETERMINATION OF CYTOTOXICITY
- The percentage of cells with structural or numerical aberrations were measured.
- To insure the evaluation of first division metaphase cells, the dividing cells were arrested in metaphase and harvested for microscopic evaluation of chromosome aberrations at approximately 24 hours after initiating treatment.

Evaluation criteria:

The solvent and positive controls fulfilled the requirements for a valid test.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY ASSAY
- Visible precipitate was observed in test medium concentrations of 312.5 to 5000 µg/mL. The results of the preliminary toxicity test can be seen in Tables 1, 2 and 3.

MAIN TEST
- 3 Hour Treatment without Metabolic Activation
When the cells were treated for 3 hours without metabolic activation with the test material, the chromosome aberrations were less than 5%. The percentage of cells with structural or numerical aberrations in the test material treated group was not significantly increased above that of the solvent control at any dose level (p>0.05, x2. Test). The percentage of cells with structural aberrations in the Mitomycin C (positive control) group was statistically significant (p< 0.01, x2 Test).

- 24 Hour Treatment without Metabolic Activation
When the cells were treated for 24 hours without metabolic activation, the test material did not cause the chromosome aberration rate to be more than 5%. The percentage of cells with structural or numerical aberrations in the test material treated group was not significantly increased above that of the solvent control at any dose level (p > 0.05, x2. Test). The percentage of cells with structurally aberrations in the Mitomycin C (positive control) group was statistically significant (p<0.01, x2 Test).

- 3 Hour Treatment with Metabolic Activation
When the cells were treated for 3 hours with metabolic activation, the test material did not cause the chromosome aberration rate to be more than 5%. The percentage of cells with structural or numerical aberrations in the test material treated group was not significantly increased above that of the solvent control at any dose level (p>0.05, x2 Test). The percentage of cells with structurally aberrations in the Cyclophosphamide (positive control) group was statistically significant (p<0.01, x2 Test).

Table 1: Preliminary toxicity assay (in the non-activated 3 hour exposure group)

Concentration (µg/mL)	5000	2500	1250	625	312.5	156.2	78.1	39.0	19.5	9.75	0 (DMSO)
Cell survival rate (%)	0	9.7	36.3	74.3	86.6	94.6	95.0	95.7	100.2	97.4	100.0
Solubility of the test material	+	+	+	+	+	-	-	-	-	-	-

 

Table 2: Preliminary toxicity assay (in the non-activated 24 hour exposure group)

Concentration (µg/mL)	5000	2500	1250	625	312.5	156.2	78.1	39.0	19.5	9.75	0 (DMSO)
Cell survival rate (%)	0	9.1	20.7	43.7	60.6	81.7	99.6	102.2	99.1	97.1	100.0
Solubility of the test material	+	+	+	+	+	-	-	-	-	-	-

 

Table 3: Preliminary toxicity assay (in the S9-activated 3 hour exposure group)

Concentration (µg/mL)	5000	2500	1250	625	312.5	156.2	78.1	39.0	19.5	9.75	0 (DMSO)
Cell survival rate (%)	0	6.8	17.9	31.8	40.0	60.3	97.8	96.7	100.4	101.3	100.0
Solubility of the test material	+	+	+	+	+	-	-	-	-	-	-

 

+ = visible precipitate, - = solution

Table 4: The results of the in vitro chromosome aberration assay of the test material on cultured CHL cells

Treatment	Dose (µg/mL)	S9 activation	Treatment time (h)	Counting cells	Number of aberrations		Rate of aberrations (%)
					Numerical	Structural	Numerical	Structural
DMSO	0	-	3	200	0	2	0	1.0
Mitomycin C	0.25	-	3	100	0	51	0	51.0**
Test material	320	-	3	200	0	3	0	1.5
	160	-	3	200	0	0	0	0.0
	80	-	3	200	0	2	0	1.0
DMSO	0	-	24	200	0	1	0	0.5
Mitomycin C	0.20	-	24	100	0	46	0	46.0**
Test material	320	-	24	200	0	1	0	0.5
	160	-	24	200	0	4	0	2.0
	80	-	24	200	0	2	0	1.0
DMSO	0	+	3	200	0	1	0	0.5
Cyclophosphamide	20	+	3	100	0	43	0	43.0**
Test material	240	+	3	200	0	2	0	1.0
	120	+	3	200	0	1	0	0.5
	60	+	3	200	0	2	0	1.0

 

** = P < 0.01, compared with DMSO control

Conclusions:

Under the conditions of the study, the test material was concluded to be negative for the induction of structural or numerical chromosome aberrations in both non-activated and S9 activated test systems in the in vitro mammalian chromosome aberration test using CHL cells. The test material was not considered to be clastogenic.

Executive summary:

The genetic toxicity of the test material was examined using the chromosome aberration test in accordance with the standardised guidelines OECD 473, EPA OPPTS 870.5375 and "Guidelines for the testing of chemicals Section 4: Health Effects, Ministry of environmental protection of People's Republic of China, 473". The purpose of this study was to evaluate the clastogenic potential of the test material based on its ability to induce chromosome aberration in Chinese hamster lung fibroblasts (CHL) cells. The study consisted of a preliminary toxicity assay and a chromosome aberration assay. The clastogenic potential of the test material was evaluated by measuring the frequency of cells with structural chromosome aberrations in CHL cultures treated with the test material in comparison with frequency in cultures treated with the solvent only. The test material was also assessed for its potential to induce numerical chromosome aberrations. The chromosome aberration assay was conducted using standard procedures by exposing CHL cells to various concentrations of test material in the presence and absence of exogenous metabolic activation supplied by Aroclor-induced rat liver S9. DMSO was used as solvent for the test material and was the solvent control. In the non-activated test system, treatment time was 3 hours or 24 hours; and in the S9 activated system the treatment time was 3 hours. To insure the evaluation of first division metaphase cells, the dividing cells were arrested in metaphase and harvested for microscopic evaluation of chromosome aberrations at approximately 24 hours after initiating treatment. In the non-activated 3 hour and 24 hour exposure groups, CHL cultures were exposed to the test material at concentrations of 320, 160 and 80 µg/mL. In the S9 activated 3 hour exposure group, CHL cultures were exposed to the test material at concentrations of 240, 120 and 60 µg/mL. Solvent control (DMSO) and appropriate positive controls (Mitomycin C in the absence of S9 and Cyclophosphamide in the presence of S9) also were tested for each treatment condition.

For each of the exposure groups, there was no statistically significant increase in the percentage of cells with structural or numerical chromosome aberrations compared with the corresponding solvent control at any of the test material concentrations evaluated microscopically. The solvent and positive controls fulfilled the requirements for a valid test. Under the conditions of the study the test material was concluded to be negative for the induction of structural or numerical chromosome aberrations in both non-activated and S9 activated test systems in the in vitro mammalian chromosome aberration test using CHL cells. The test material was not considered to be clastogenic in this in vitro study.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 August 2009 to 21 September 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidelines for Testing of Toxic Chemicals, Ministry of Health, People's Republic of China, 272.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidelines for the testing of chemicals Section 4: Health Effects, Ministry of environmental protection of People's Republic of China, 471.

Version / remarks:

2003

Deviations:

no

GLP compliance:

not specified

Remarks:

Not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

Species / strain / cell type:

S. typhimurium, other: TA 97, 98, 100 and 102.

Details on mammalian cell type (if applicable):

CELLS USED
The R-factor strains (TA97, TA98, TA100 and TA102) were tested for the presence of ampicillin resistance factor. The pAQ1 strain (TA102) was tested for both ampicillin and tetracycline resistance on ampicillin/tetracycline plates. Spontaneous reversion was also measured with routine procedures. The shaker/incubator was programmed to begin shaking at 100 rpm at 37°C for 10 hours before the anticipated time of harvest. Each culture was cultivated to increase the final bacterial concentration up to 3 x10^8 bacteria/mL.

MEDIA USED
- Top agar, bottom agar and nutrient broth were prepared according to Ames assay.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

- 0.5, 5, 50, 500 and 5000 µg per plate concentrations were used for the preliminary toxicity and mutagenicity assay.
- Precipitate wasn't observed at any dose level. No appreciable toxicity was observed at 5000, 500, 50, 5 or 0.5 µg per plate. Based on the findings of the preliminary toxicity assay, the maximum dose plated in the mutagenicity assay was 5000 µg per plate. The mutagenicity assay used the same concentrations as the preliminary study.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Dimethyl sulfoxide (DMSO) was selected as the solvent based on the compatibility with the target test strains. All positive control articles were diluted with DMSO except sodium azide, which was diluted with water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-aminofluoresce, Dexon and 1,8-dioxyanthraquinone

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and pre-incubation.
- The initial mutagenicity assays was conducted using the plate incorporation method. The confirmatory mutagenicity assay was conducted using the preincubation method.
- In the plate incorporation method, 0.5 mL of S9 or Sham mix, 100 µL of test stain and 100 µL of vehicle or test material dilution were added to 2.0 mL of molten selective top agar at 45°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar.
- In the pre-incubation method, 0.5 mL of S9 or sham mix, 100 µL of test stain and 100 µL of vehicle or test material dilution were added to glass culture tubes pre-heated to 37°C. After vortexing, these mixtures were incubated for 20 minutes at 37°C. Following the pre-incubation, 2.0 mL of selective top agar was added to each tube and the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 100 µL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 hours at 37°C.
- The condition of the bacterial background lawn was evaluated for evidence of test material toxicity by using a magnifier. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate.

CRITERIA FOR A VALID TEST
- All Salmonella test strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene (except TA102). Cultures of test strains TA97, TA98, and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls. To ensure that appropriate numbers of bacteria are plated, test strain culture titers must be greater than or equal to 3 x 10^8 cells/mL. The mean of each positive control must exhibit at least two fold increase in the number of revertants over the mean value of the respective vehicle control. A minimum of three non-toxic dose levels is required to evaluate assay data.
- A dose level is considered toxic if one or both of the following criteria are met: (1) A >50% reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by dose-dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn (background lawn code 3, 4 or 5)

Evaluation criteria:

EVALUATION OF RESULTS
For the test material to be considered as positive, it must cause a dose-related increase in the mean revertants per plate of at least one test strain over a minimum of two increasing concentrations of test material. Data sets for test strains TA97, TA98, TA100 and TA102 were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than two times the mean vehicle control value.

Key result

Species / strain:

S. typhimurium, other: TA97, TA98, TA100 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

SOLUBILITY
The test material was soluble in DMSO at a concentration of 50 mg/mL, the maximum concentration prepared for this study.

STERILITY RESULTS
No contaminant colonies were observed on the sterility plates for the vehicle, the test material dilutions, the S9 and Sham mixes.

PRELIMINARY TOXICITY ASSAY
The maximum dose tested was 5000 µg per plate; this dose was achieved using a concentration of 50 mg/mL and a 100 µL plating aliquot. The dose levels tested were 0.5, 5, 50, 500 and 5000 µg per plate. Precipitate wasn't observed at all dose levels. No appreciable toxicity was observed at 5000, 500, 50, 5 or 0.5 µg per plate. Based on the findings of the preliminary toxicity assay, the maximum dose plated in the mutagenicity assay was 5000 µg per plate.

MUTAGENICITY ASSAY
The results of the mutagenicity assay are presented in Tables 1 and 2; these data were generated in experiments B1 and B2.
- In experiment B1 (initial mutagenicity assay via plate incorporation), no positive mutagenicity responses were observed with any of the test strains in either the presence or absence of S9 activation. Precipitate wasn't observed at any dose levels. At all dose levels significant toxicity was not observed. The background bacterial lawn cannot be accurately evaluated at 5000 µg per plate due to microscopic test article particulate.

- In experiment B2 (confirmatory mutagenicity assay via preincubation), no positive mutagenicity responses were observed with any of the test strains in either the presence or absence of S9 activation. Precipitate was not observed at any dose levels. Significant toxicity was not observed at any dose level. The background bacterial lawn cannot be accurately evaluated at 5000 µg per plate due to microscopic test article particulate.

Table 1: Summary of the Initial Mutagenicity test

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		TA97	TA98	TA100	TA102
-	Solvent (H2O) Solvent (DMSO) 0.5 5 50 500 5000	108 ± 13 109 ± 11 127 ± 9 112 ± 5 115 ± 10 119 ± 16 109 ± 9	34 ± 3 32 ± 2 32 ± 3 33 ± 6 36 ± 3 39 ± 5 33 ± 3	162 ± 20 176 ± 21 173 ± 16 160 ± 13 169 ± 24 175 ± 18 177 ± 19	257 ± 16 270 ± 10 286 ± 14 291 ± 10 269 ± 30 275 ± 25 270 ± 22
+	Solvent (H2O) Solvent (DMSO) 0.5 5 50 500 5000	124 ± 9 118 ± 4 113 ± 8 121 ± 15 103 ± 12 120 ± 12 117 ± 14	38 ± 6 37 ± 4 35 ± 2 38 ± 3 36 ± 1 35 ± 4 34 ± 3	176 ± 15 158 ± 22 183 ± 4 163 ± 19 160 ± 7 160 ± 11 154 ¿1 18	263 ± 19 274 ± 8 263 ± 25 253 ± 15 282 ± 21 284 ± 10 275 ± 12
Positive Controls
-	Name	Dexon	Dexon	SA	Dexon
	Concentration (µg/plate)	50	50	1.5	50
	Mean no. colonies/plate	1970 ± 200	1344 ± 162	1986 ± 173	1083 ¿1 175
+	Name	2-AF	2-AF	2-AF	1,8-dioxyant hraquinone
	Concentration (µg/plate)	10	10	10	50
	Mean no. colonies/plate	1166 ± 109	3575 ± 283	2716 ± 218	1171 ± 118

2-AF = 2-aminofluoresce

SA = Sodium azide

Table 2: Summary of the Confirmatory Mutagenicity test

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		TA97	TA98	TA100	TA102
-	Solvent (H2O) Solvent (DMSO) 0.5 5 50 500 5000	122 ± 14 107 ± 14 110 ± 5 123 ± 12 128 ± 7 119 ± 14 122 ± 11	37 ± 3 37 ± 5 39 ± 4 36 ± 4 37 ± 5 33 ± 3 32 ± 3	173 ± 16 156 ± 14 155 ± 16 167 ± 23 164 ± 11 150 ± 10 174 ± 8	274 ± 22 261 ± 18 257 ± 9 251 ± 8 278 ± 27 276 ± 20 293 ± 16
+	Solvent (H2O) Solvent (DMSO) 0.5 5 50 500 5000	110 ± 10 114 ± 11 115 ± 7 110 ± 10 106 ± 9 117 ± 18 131 ± 10	31 ± 2 34 ± 2 37 ± 6 35 ± 4 33 ± 2 34 ± 2 35 ± 4	152 ± 11 182 ± 13 169 ± 24 176 ± 18 180 ± 12 157 ± 13 190 ± 7	263 ± 9 290 ± 9 277 ± 19 283 ± 29 276 ± 11 279 ± 23 264 ± 19
Positive Controls
-	Name	Dexon	Dexon	SA	Dexon
	Concentration (µg/plate)	50	50	1.5	50
	Mean no. colonies/plate	1941 ± 127	1263 ± 137	2057 ± 131	1060 ± 105
+	Name	2-AF	2-AF	2-AF	1,8-dioxyant hraquinone
	Concentration (µg/plate)	10	10	10	50
	Mean no. colonies/plate	1154 ± 133	3500 ± 174	2796 ± 133	1051± 146

Conclusions:

Under the conditions of the study the test material was not mutagenic with Salmonella typhimurium test strains TA97, TA98, TA100 and TA102 in either the presence or absence of S9 metabolic activation.

Executive summary:

The genetic toxicity of the test material was determined in accordance with the standardised guidelines OECD 471, EPA OPPTS 870.5100 and "The Guidelines for the Testing of Chemicals Section 4: Health Effects (ministry of environmental protection of People's Republic of China" using a reverse mutation assay. The purpose of this study was to evaluate the mutagenic potential of the test material, by testing its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium test strains in the presence and absence of S9 activation. DMSO served as the vehicle control; the vehicle control and positive controls (Dexon, Sodium azide, 1,8-dioxyanthraquinone, 2-AF) were included in the assays. Test strains were Salmonella strains TA97, TA98, TA100 and TA102. All dose levels of test material, vehicle control and positive controls were performed in triplicate. In both initial and confirmatory mutagenicity assays, the test material was tested in the presence and absence of Aroclor-induced rat liver S9 metabolic activation at 0.5, 5, 50, 500 and 5000 µg per plate. The initial mutagenicity assay was conducted using the plate incorporation method. The confirmatory mutagenicity assay was conducted using the preincubation method. In all trials, no appreciable toxicity was evident up to 5000 µg per plate with the test material in the presence and absence of S9 metabolic activation. In the initial and confirmatory mutagenicity assays, precipitate wasn't observed at 0.5, 5, 50, 500 and 5000 µg per plate but the background bacterial lawn cannot be accurately evaluated at 5000 µg per plate due to microscopic test material particulate. No positive mutagenic response was observed. The vehicle control and positive controls fulfilled the requirements for a valid assay. Under the conditions of the study the test material was not mutagenic with Salmonella typhimurium test strains TA97, TA98, TA100 and TA102 in either the presence or absence of S9 metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Chromosome aberration

The genetic toxicity of the test material was examined using the chromosome aberration test in accordance with the standardised guidelines OECD 473,EPA OPPTS 870.5375 and "Guidelines for the testing of chemicals Section 4: Health Effects, Ministry of environmental protection of People's Republic of China, 473".The purpose of this study was to evaluate the clastogenic potential of the test material based on its ability to induce chromosome aberration in Chinese hamster lung fibroblasts (CHL) cells. The study consisted of a preliminary toxicity assay and a chromosome aberration assay. The clastogenic potential of the test material was evaluated by measuring the frequency of cells with structural chromosome aberrations in CHL cultures treated with the test material in comparison with frequency in cultures treated with the solvent only. The test material was also assessed for its potential to induce numerical chromosome aberrations. The chromosome aberration assay was conducted using standard procedures by exposing CHL cells to various concentrations of test material in the presence and absence of exogenous metabolic activation supplied by Aroclor-induced rat liver S9. DMSO was used as solvent for the test material and was the solvent control. In the non-activated test system, treatment time was 3 hours or 24 hours; and in the S9 activated system the treatment time was 3 hours. To insure the evaluation of first division metaphase cells, the dividing cells were arrested in metaphase and harvested for microscopic evaluation of chromosome aberrations at approximately 24 hours after initiating treatment. In the non-activated 3 hour and 24 hour exposure groups, CHL cultures were exposed to the test material at concentrations of 320, 160 and 80 µg/mL. In the S9 activated 3 hour exposure group, CHL cultures were exposed to the test material at concentrations of 240, 120 and 60 µg/mL. Solvent control (DMSO) and appropriate positive controls (Mitomycin C in the absence of S9 and Cyclophosphamide in the presence of S9) also were tested for each treatment condition.

For each of the exposure groups, there was no statistically significant increase in the percentage of cells with structural or numerical chromosome aberrations compared with the corresponding solvent control at any of the test material concentrations evaluated microscopically. The solvent and positive controls fulfilled the requirements for a valid test. Under the conditions of the study the test material was concluded to be negative for the induction of structural or numerical chromosome aberrations in both non-activated and S9 activated test systems in the in vitro mammalian chromosome aberration test using CHL cells. The test material was not considered to be clastogenic in this in vitro study.

Ames Test

The genetic toxicity of the test material was determined in accordance with the standardised guidelines OECD 471, EPA OPPTS 870.5100 and "The Guidelines for the Testing of Chemicals Section 4: Health Effects (ministry of environmental protection of People's Republic of China" using a reverse mutation assay. The purpose of this study was to evaluate the mutagenic potential of the test material, by testing its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium test strains in the presence and absence of S9 activation. DMSO served as the vehicle control; the vehicle control and positive controls (Dexon, Sodium azide, 1,8-dioxyanthraquinone, 2-AF) were included in the assays. Test strains were Salmonella strains TA97, TA98, TA100 and TA102. All dose levels of test material, vehicle control and positive controls were performed in triplicate. In both initial and confirmatory mutagenicity assays, the test material was tested in the presence and absence of Aroclor-induced rat liver S9 metabolic activation at 0.5, 5, 50, 500 and 5000 µg per plate. The initial mutagenicity assay was conducted using the plate incorporation method. The confirmatory mutagenicity assay was conducted using the preincubation method. In all trials, no appreciable toxicity was evident up to 5000 µg per plate with the test material in the presence and absence of S9 metabolic activation. In the initial and confirmatory mutagenicity assays, precipitate wasn't observed at 0.5, 5, 50, 500 and 5000 µg per plate but the background bacterial lawn cannot be accurately evaluated at 5000 µg per plate due to microscopic test material particulate. No positive mutagenic response was observed. The vehicle control and positive controls fulfilled the requirements for a valid assay. Under the conditions of the study the test material was not mutagenic with Salmonella typhimurium test strains TA97, TA98, TA100 and TA102 in either the presence or absence of S9 metabolic activation.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.