1-Ethoxy-2,3-difluoro-4-[trans-4-(trans-4-propylcyclohexyl)-cyclohexyl]-benzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic in a bacterial reverse mutation assay.

The test material was non-mutagenic in mouse lymphoma cells in the absence or presence of S9 mix.

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:

Apr 28 - Jul 07, 1997

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

HIS operon (S. thyphimurium)
TRP operon (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

his C 3076, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

his D 3052, uvrB, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

his G 428, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

E. coli WP2

Details on mammalian cell type (if applicable):

uvrA pkM101

Additional strain / cell type characteristics:

other: mutations in the tryptophan operon

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors

Test concentrations with justification for top dose:

The test material concentrations used were selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan:
1. Series: 1.58, 5, 15.8, 50, and 158 µg/plate (S9 10 %)
1. Series: 1.58, 5, 15.8, 50, and 158 µg/plate (S9 30 %)

Vehicle / solvent:

acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

cumene hydroperoxide

other: daunomycin

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

with S9

Details on test system and experimental conditions:

The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. The following criteria, based upon the historical controls of the laboratory and statistical considerations, are established:

-----------------------------------------------------------------------------------------
Mean Number of Colonies Maximal Mean Number of Colonies over the Actual
(Solvent Control) Solvent Control
(Test Material)
-----------------------------------------------------------------------------------------

<=10 <=9 >=30
<=30 <=19 >=40
<=80 <=29 >=80
<=200 <=49 >=120
<=500 <=79 >=200
Assessment: No increase Clear increase

-----------------------------------------------------------------------------------------

All further results, ranging between "no" and "clear", are assessed as "weak increases".

Interpretations:
A test material is defined as non-mutagenic in this assay if:

- "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)

A test material is defined as mutagenic in this assay if:

- a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;

- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

In all further cases, a third test series with the bacterial strain in question should be performed.
If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.

Evaluation criteria:

Please refer to "Details on test system and experimental conditions".

Statistics:

n.a.

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation was observed at 158 µg/plate (without S9 at end of exposure, with S9 at beginning of exposure).

STUDY RESULTS
Please refer to attachment for additional information on results.

Conclusions:

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic in a bacterial reverse mutation assay.

Executive summary:

The purpose of this assay was to provide information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man. The investigations for the mutagenic potential of the test material were performed according to OECD 471 using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535, TA 1537 and Escherichia coli WP2 uvrA pKM101. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. The test material was dissolved in acetone and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at the highest concentration tested (158 µg/plate). Toxicity to the bacteria was not observed. Each treatment with the positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. According to the criteria for negative and positive results, the test material was not mutagenic under the described experimental conditions. With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 June - 24 November 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

21st July 1997

Deviations:

yes

Remarks:

less concentrations, only single cultures per concentration, and a simplified data reporting

Qualifier:

according to guideline

Guideline:

other: EEC Directive 79-831

Version / remarks:

Annex V, EEC Directive 79-831, Part B, Toxicological Methods of Annex VIII. In vitro Mammalian cell - gene mutation test.
Commission of the European Communities, March 1985:85-7.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspects of Regulatory Tests (1995) and ICH Requirements for Registration of Pharmaceuticals for Human Use, Geno¬toxicity: a Standard Battery for Genotoxicity Testing of Pharmaceuticals (Step 4, recommended for adoption 16 July 1997).

Principles of method if other than guideline:

The methodology, originally complying with the ICH guidelines, OECD Test Guideline 476 and Annex V, EEC Directive 79-831, was modified in this study in order to screen a large number of test materials. In brief, the main modification is the use of fewer concentrations of the test material, use of only single cultures per concentration and a simplified data reporting.

GLP compliance:

no

Type of assay:

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

Target gene:

gene locus of thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from livers of rats pretreated with Aroclor 1254

Test concentrations with justification for top dose:

In a preceding range finding test, cell growth was determined after exposure (3 hours in the presence and 24 hours in the absence of S9 mix) to various test material concentrations. The cell number was determined microscopically 24 hours after start of treatment and compared with cell number in the absence of the test material. The test material was weakly cytotoxic (67 % cell growth at 28.1 µg/mL in the presence of S9 mix as compared to the actual solvent control) in this range finding test.

Precipitation of the test material in the cell culture medium was macroscopically visible at concentrations >- 28.1 µg/mL.

24 hours treatment without S9 mix: 8.89, 15.8 and 28.1 µg /mL
3 hours treatment with S9 mix: 8.89, 15.8 and 28.1 µg /mL

Vehicle / solvent:

Acetone (0.1 % (v/v)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 24 hours without metabolic activation, 3 hours with metabolic activation
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 5-trifluorothymidine

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: >1x10e6

DETERMINATION OF CYTOTOXICITY
- Method: relative survival

Evaluation criteria:

Test materials are assessed as negative or non-mutagenic in this test system if
• the assay is considered valid and
• no relevant increase in the mutation frequency (at least a 2-fold) occurs.

Test materials are assessed as positive or mutagenic in this test system if
• the assay is considered valid and
• a clear increase in the mutation frequency (at least a 2-fold) occurs dose-dependently (over at least two test material concentrations) or reproducibly at identical concentrations in two independent experimental series performed.

In all other cases, further decisions for testing strategies should be made following the scientific evaluation of all existing data including those of non-toxicological investigations.

Statistics:

No statistics was applied, biological significance was considered as main factor for evaluation.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: determined at 28.1 µg/mL
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
In a preceding range finding test, cell growth was determined after exposure (3 hours in the presence and 24 hours in the absence of S9 mix) to various test material concentrations. The cell number was determined microscopically 24 hours after start of treatment and compared with cell number in the absence of the test material. The test material was weakly cytotoxic (67 % cell growth at 28.1 µg/mL in the presence of S9 mix as compared to the actual solvent control) in this range finding test.

Precipitation of the test material in the cell culture medium was macroscopically visible at concentrations of 28.1 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA: Complies

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effect indicated by an adjusted relative survival below 50 % in both cultures occurred up to the maximum concentration with and without metabolic activation.

 

Without S9 mix (EZ1144):

Test Material	Conc. [µg/mL]	Rel.Survival [%]a	Mutation frequ.b
Solvent		100	253
			172
Test item	8.89	69	228
	15.8	82	154
	28.1^PP	77	102
NQO	0.10	43	776
	0.20	56	706

 

 

 

With S9 mix(EZ1144):

Test Material	Conc. [µg/mL]	Rel.Survival [%] a	Mutation frequ. b
Solvent		100	188
			202
Test item	8.89	284	180
	15.8	230	137
	28.1^PP	189	238
BaP	2.00	230	987
	3.00	218	755

a: On day 1 of the experiment                                b: Per 10⁶ viable cells

NQO: 4-Nitroquinoline N-oxide;                           BaP: Benzo[a]pyrene

PP: Test material precipitated until end of the exposure time

Conclusions:

The test material was non-mutagenic in mouse lymphoma cells in the absence or presence of S9 mix.

Executive summary:

In a study conducted according to OECD 476 the test material was screened for its ability to induce mutations at the TK locus (5-tri-fluorothymidine (TFT) resistance) in mouse lymphoma cells using a fluctuation protocol. The study was conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254). The exposure time was 24 hours in the absence and 3 hours in the presence of S9 mix. The top doses to determine TFT resistance in this experiment were selected on the basis of the solubility and cytotoxicity characteristics of the test material. The test material precipitated in the culture medium at concentrations exceeding 28.1 µg/mL. The test material was non-toxic to the mouse lymphoma cells in the absence and presence of S9 mix. Concentrations ranging from 8.89 to 28.1 µg/mL were therefore tested. Negative (solvent) and positive control treatments were included in each mutation experiment in the absence and presence of S9 mix. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline N-oxide (without S9 mix) and benzo[a]pyrene (with S9 mix). Therefore, the study was accepted as valid. No relevant increases in mutant frequency were observed following treatment with the test material in either the absence or presence of S9 mix. It is therefore concluded that the test material is non-mutagenic in this screening test system under conditions where the positive controls exerted potent mutagenic effects.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

OECD 471

The purpose of this assay was to provide information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man. The investigations for the mutagenic potential of the test material were performed according to OECD 471 using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535, TA 1537 and Escherichia coli WP2 uvrA pKM101. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. The test material was dissolved in acetone and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at the highest concentration tested (158 µg/plate). Toxicity to the bacteria was not observed. Each treatment with the positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. According to the criteria for negative and positive results, the test material was not mutagenic under the described experimental conditions. With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

 

OECD 476

In a study conducted according to OECD 476 the test material was screened for its ability to induce mutations at the TK locus (5-tri-fluorothymidine (TFT) resistance) in mouse lymphoma cells using a fluctuation protocol. The study was conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254). The exposure time was 24 hours in the absence and 3 hours in the presence of S9 mix. The top doses to determine TFT resistance in this experiment were selected on the basis of the solubility and cytotoxicity characteristics of the test material. The test material precipitated in the culture medium at concentrations exceeding 28.1 µg/mL. The test material was non-toxic to the mouse lymphoma cells in the absence and presence of S9 mix. Concentrations ranging from 8.89 to 28.1 µg/mL were therefore tested. Negative (solvent) and positive control treatments were included in each mutation experiment in the absence and presence of S9 mix. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline N-oxide (without S9 mix) and benzo[a]pyrene (with S9 mix). Therefore, the study was accepted as valid. No relevant increases in mutant frequency were observed following treatment with the test material in either the absence or presence of S9 mix. It is therefore concluded that the test material is non-mutagenic in this screening test system under conditions where the positive controls exerted potent mutagenic effects.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity in vitro, the test item doesnot require classification according to Regulation (EC) No 1272/2008 (CLP).