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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 August 2009 to 21 September 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

946-937-7

Molecular formula:

C36H62N2O8

IUPAC Name:

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

Test material form:

liquid

Details on test material:

- Storage: cool and dark

Method

Species / strain

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

Controls

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.

Results and discussion

Test results

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).

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.