Bis[2-[2-(1-methylethyl)-3-oxazolidinyl]ethyl] hexan-1,2-diylbiscarbamate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-09-25 to 2012-12-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

Bis[2-[2-(1-methylethyl)-3-oxazolidinyl]ethyl] hexan-1,2-diylbiscarbamate

EC Number:

261-879-6

EC Name:

Bis[2-[2-(1-methylethyl)-3-oxazolidinyl]ethyl] hexan-1,2-diylbiscarbamate

Cas Number:

59719-67-4

Molecular formula:

C24H46N4O6

IUPAC Name:

bis[2-(2-isopropyl-1,3-oxazolidin-3-yl)ethyl] hexane-1,6-diylbiscarbamate

Method

Target gene:

tk+/- (thymidine kinase)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 mediumcontaining 25 mM HEPES and 2 g/L NaHCO3 and 0.3 g/L L-Glutamine
RPMI 5: 5 % (v/v) Horse serum (heat inactivated), 0.01 mL/mL Antibiotic-Antimycotic solution, 0.5 mg/mL Pluronic-F68, 0.2 mg/mL Pyruvic acid
RPMI 10: 10 % (v/v) Horse serum (heat inactivated), 0.01 mL/mL Antibiotic-Antimycotic solution, 0.5 mg/mL Pluronic-F68, 0.2 mg/mL Pyruvic acid
RPMI 20: 20 % (v/v) Horse serum (heat inactivated), 0.01 mL/mL Antibiotic-Antimycotic solution, 0.2 mg/mL Pyruvic acid
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver (provided by Trinova Biochem GmbH) and cofactors

Test concentrations with justification for top dose:

Assay 1:
3-hour treatment, -S9: 100; 500; 1000; 1500; 2000; 2500 and 3000 μg/mL
3-hour treatment, +S9: 100; 500; 1000; 1500; 1750; 2000 and 2500 μg/mL
Assay 2:
24-hour treatment, -S9 Mix: 100; 200; 300; 400; 500; 600 and 750 μg/mL
3-hour treatment, +S9 Mix: 500; 1000; 1500; 1750; 2000 and 2500 μg/mL

Vehicle / solvent:

- Vehicle used: Abs. Ethanol
- Justification for choice of vehicle: The test item was solubile in the vehicle up to a concentration of 100 mg/mL. The vehicle has no deleterious or mutagenic effect on the test system.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Assay 1: 3 hours with and without metabolic activation; Assay 2: 3 hours with and 24 hours without metabolic activation
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 - 14 days

SELECTION AGENT (mutation assays): 5-trifluorothymidine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER: pH and Osmolality

Evaluation criteria:

Assay Acceptance Criteria
The assays are considered valid if all of the following criteria are met:
1. The mutant frequency in the negative (vehicle) control cultures fall within the normal range (above 50 - 170 mutants per 106 viable cells).
2. The positive control chemicals induce a statistically significant increase in the mutant frequency.
3. The plating efficiency (PEviability) of the negative controls is within the range of 65 % to 120 % on Day 2 (at the end of the expression period).
4. At least four test concentrations are present, where the highest concentration produces 80 - 90 % toxicity, precipitation, or is 5 mg/mL, or the highest practical concentration.

Mutagenicity Criteria
The test item is considered to be mutagenic in this assay if all the following criteria are met:
1. The assay is valid.
2. Statistically significant (p < 0.05) increases in mutation frequency are observed in treated cultures compared to the corresponding negative control values at one or more concentrations.
3. The increases are reproducible between replicate cultures and between tests.
4. There is a significant dose-relationship as indicated by the adequate trend analysis.
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 1E-6 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative control value.

Statistics:

For statistical evaluation of mutation frequencies of the test item concentrations were tested on significant differences to the control values by Dunnett’s Test at the positive controls by 2 Sample t-Test by TOXSTAT software. The data were checked for normality by Chi-Square Test, for homogeneity of variance for Bartlett’s Test.

Results and discussion

Test results

Key result

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

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: yes

RANGE-FINDING STUDIES: Yes, the obtained survival data were used to choose the dose levels for the main assays with the aim of covering a concentration range from the maximum possible cytotoxicity to a low effect level.

COMPARISON WITH HISTORICAL CONTROL DATA: In the Assay 1 as well as in the Assay 2 the plating efficiencies of the negative and positive controls in the viability test (PEviability) as well as the mutation frequency of the current negative control were within the acceptable ranges and in accordance with the historical data.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The examined concentrations covered the cytotoxicity range (based on the harmonised relative survival and/or relative total growth) from the 10 - 20 % cytotoxicity to no toxicity. To fulfill the cytotoxicity criterion of the assay, the test item was investigated beyond its limit of solubility under culture conditions.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test item Incozol 4 does not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells used.

Executive summary:

An in vitro mammalian cell gene mutation assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of the test item to cause gene mutation and/or chromosome damage. Treatments were carried out for 3 hours with and without metabolic activation (±S9 Mix) and for 24 hours without metabolic activation (-S9 Mix).

The appropriate vehicle, absolute ethanol (Abs. Ethanol) was chosen based on the results of the preliminary solubility test. The test item solutions were prepared in Abs. Ethanol and diluted prior to treatment. Based on the available historical control database, this vehicle was compatible with the survival of the cells and the S9 activity. The test item was tested beyond its limit of solubility under culture conditions.

The concentrations applied in the Assay 1 were chosen according to the solubility and cytotoxicity results of the pre-experiments.

The following concentrations were investigated in the Assay 1:

3-hour treatment (-S9 Mix): 100; 500; 1000; 1500; 2000; 2500 and 3000 μg/mL;

3-hour treatment (+S9 Mix): 100; 500; 1000; 1500; 1750; 2000 and 2500 μg/mL.

Concentrations investigated in the Assay 2:

24-hour treatment (-S9 Mix): 100; 200; 300; 400; 500; 600 and 750 μg/mL;

3-hour treatment (+S9 Mix): 500; 1000; 1500; 1750; 2000 and 2500 μg/mL.

In the performed mutation assays the cell cultures were treated with a range of the test item concentrations. After the treatment the cell cultures were washed, re-suspended, the cell densities determined and adjusted to 2x105/mL. The cells were transferred to flasks for growth through the expression period (for approximately 2 days) or diluted to be plated for survival. At the end of the expression period cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT) for determination of mutations and viability.

The performed Assays fulfilled the validity criteria in connection with the negative control and positive control treatments.

In the performed Assay 1 and Assay 2 in the absence and also in presence of S9 Mix dose-related toxicity of the test item was observed based on both, the harmonised relative survival (harmonised RS) and the relative total growth (RTG) values. The concentration levels of 2500 and 3000 μg/mL in the Assay 1 and the concentration level of 750 μg/mL in the Assay 2 in absence of exogenous metabolic activation were out of the analysable range (higher than 90 % cytotoxicity occurred at these concentration levels). Apart from this the guideline criteria for the number of analysable concentration levels (at least four) as well as the required cytotoxicity degree (10 - 20 % relative survival or relative total growth) at the maximum concentration (in absence of metabolic activation the maximum analyzable concentration) were fulfilled. In presence of metabolic activation the cytotoxicity caused by the highest examined concentration level was in the acceptable 80 - 90 % range in both assays.

Significant differences to the concurrent control were noted for some test concentrations (causing high toxicity down to the limit described in the guideline) in the first and second experiment (Dunnett’s Test, α= 0.05 or α= 0.01). No clear concentration-response was observed in either of the tests with and without metabolic activation. Apart from these findings the obtained mutation frequencies remained unequivocally well below the GEF criterion for positive response in all cases. The final conclusion was drawn from the results of the statistical and biological evaluation.

Under the conditions of this study, the test item does not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells used.