1-Ethoxy-2,3-difluoro-4-(trans-4-propylcyclohexyl)-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 bacteria.

The test material did not induce structural or numerical chromosome aberrations in cultured Chinese hamster ovary (CHO) cells when tested to its limit of cytotoxicity in both the absence and presence of metabolic activation (S-9).

The test material did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of solubility, for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9).

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:

March 12 - May 28, 1997

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

HIS operon (S. thyphimurium)
TRY 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):

his C 3076, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the tryptophan operon

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate (S9 mix) with standard co-factors with metabolic activation (Aroclor)

Test concentrations with justification for top dose:

The test material concentrations were used selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan: 1. Series: 5, 15.8, 50, 158, 500, 1580, and 5000 µg/plate and 2. Series: 15.8, 50, 158, 500, and 1580 µg/plate

Vehicle / solvent:

Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

cumene hydroperoxide

other: 2-aminoanthracene, Aminoacridine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: test item and positive control: 3; solvent control: 5
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Evaluation criteria:

The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the in-crease 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:

(A) (B)
≤10 ≤9 ≥30
≤30 ≤19 ≥40
≤80 ≤29 ≥80
≤200 ≤49 ≥120
≤500 ≤79 ≥200
Assessment: no increase clear increase
(A)=Mean Number of Colonies (Solvent Control)
(B)=Maximal Mean Number of Colonies over the Actual Solvent Control (Test Material)
All further results, ranging between "no" and "clear", are assessed as "weak in-creases".
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.

Statistics:

n.a.

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:

valid

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:

valid

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:

valid

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

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:

valid

True 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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Precipitation on agar plates: >=1580 µg/plate
Cytotoxicity: not observed

Conclusions:

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Executive summary:

Purpose

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.

 

Study Design

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 and TA 1537, and Escherichia coli WP2 uvrA. 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.

 

Results

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 concentrations larger or equal to 1580 µg/plate. Toxicity to the bacteria was not observed.

Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9 -aminoacridine and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as 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.

 

Conclusion

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 cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 17 - November 19, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy's 5A medium including 10 % (v/v) FCS
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (induction using Aroclor 1254)

Test concentrations with justification for top dose:

0, 17.75, 20.88, and 24.57 µg/mL (without S9, 20 h treatment)
0, 15.09 and 17.75 µg/mL (without S9, 44 h treatment)
0, 59.09, 65.61, and 81.00 µg/mL (with S9, 3 h treatment, 17 h recovery)
0, 65.61, and 72.90 µg/mL (with S9, 3 h treatment, 17 h recovery)

Vehicle / solvent:

Name: sterile ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: Continuous for 20 and 44 hours, Pulse for 3 + 17 and 3+41 hours
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: Controls: 4; others: 2
NUMBER OF CELLS EVALUATED: 100 metaphases (structural aberrations)
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative total growth
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A test article is considered as positive in this assay if:
1. the proportions of cells with structural aberrations at one or more concentration exceeds the normal range in both replicate cultures, and
2. a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurs at these doses.
Increased incidence of cells with gaps or increased proportions of cells with structural aberrations not exceeding the normal range, or occurring only at very high or very toxic concentrations are likely to be concluded as "equivocal". Full assessment of the biological importance of such increases is likely only to be possible with reference to data from other test systems. Evidence of a dose-related effect is considered useful but not essential in the evaluation of a positive result. Cells with exchange aberrations or cells with greater than one structural aberration occur very infrequently in negative control cultures. Their appearance is therefore considered to be of particular biological significance.

Statistics:

Standard statistical methods have been applied for data processing.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes (441 µg/mL)
- Cytotoxicity: yes
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Due to the toxicity profiles observed (reductions in cell number), it was not possible to select the same top concentrations for analysis from treatments sampled at 44 hours as those for treatments sampled at 20 hours. The pattern of toxicity for both treatments sampled at 44 hours was such that two concentration choices were possible; one inducing below 50 % cytotoxicity and one inducing 60 % (or slightly above) cytotoxicity. It was considered prudent to analyse both concentrations from both treatments. Although contrary to the protocol, this was considered to add to the data set and as such has no adverse effect on the validity of the study.

Conclusions:

It is concluded that the test material did not induce structural or numerical chromosome aberrations in cultured Chinese hamster ovary (CHO) cells when tested to its limit of cytotoxicity in both the absence and presence of metabolic activation (S-9).

Executive summary:

The test material was tested in an in vitro cytogenetics assay according to OECD 473 using duplicate cultures of Chinese hamster ovary (CHO) cells in two independent experiments. Treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9). The test article was dissolved in sterile ethanol (ethanol) and the highest dose level used, 400 μg/mL, was in excess of the solubility limit in culture medium. In Experiment 1, treatment in the absence and presence of S-9 was for 3 hours followed by a 17-hour recovery period prior to harvest (3+17). The S-9 used was prepared from a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. The test article dose levels for chromosome analysis were selected by evaluating the effect of the test material on relative cell number. Chromosome aberrations were analysed at three dose levels. The highest concentrations chosen for analysis, 27.49 and 83.89 μg/mL induced approximately 58 % and 66 % reduction in cell number and 0 % and 49 % mitotic inhibition (MIH) in the absence and presence of S-9 respectively. In Experiment 2, treatment in the absence of S-9 was continuous for either 20 hours or 44 hours. Treatment in the presence of S-9 was either for 3 hours only followed by a 17-hour recovery period prior to harvest (3+17) or for 3 hours followed by a 41-hour recovery period prior to harvest (3+41). Chromosome aberrations were analysed at two or three dose levels (see overleaf) and the highest concentrations chosen for analysis, 24.57 μg/mL (20+0) or 17.75 μg/mL (44+0) in the absence of S-9 and 81.00 μg/mL (3 +17) or 72.90 μg/mL (3+41) in the presence of S-9, induced approximately 50 %, 60 %, 54 % and 62 % reduction in cell number and 81 %, 29 %, 55 % and 7 % MIH respectively. Appropriate negative (vehicle) control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges. 4-Nitroquinoline 1 -oxide and cyclophosphamide were employed as positive control chemicals in the absence and presence of liver S-9 respectively. Cells receiving these were sampled in each experiment, 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations. Positive controls were included with both treatments in Experiment 1, but only with the 20+0 hour -S-9 and 3+17 +S-9 treatments in Experiment 2.
Treatment of cultures with the test material in the absence and the presence of S-9 (both experiments) resulted in frequencies of cells with structural aberrations which were similar to those observed in concurrent vehicle controls for the majority of concentrations analysed. Two exceptions to this were observed: a single culture at the intermediate concentration analysed (67.11 mg/mL) from the 3+17 hour +S-9 treatment in Experiment 1 and a single culture from the lowest concentration analysed (15.09 mg/mL) from the 44+0 hour –S-9 treatment in Experiment 2 both exhibited numbers of aberrant cells that exceeded historical negative control (normal) values. However, in both instances these increases were not observed in the replicate cultures and were not dose-related. Furthermore, the increase observed in the presence of S-9 in Experiment 1 was not observed at similar concentrations analysed in Experiment 2 (performed under identical treatment conditions). The aberrant cell frequency of all other test material treated cultures fell within normal values. It was therefore considered that the increases observed were spurious and of no biological significance. Normal frequencies of cells with numerical aberrations (within historical negative control (normal) ranges) were observed for the large majority of test material treated cultures. The only exception to this was observed in the 3+17 hour –S-9 treatment at the highest concentration tested (27.49 mg/mL) where a single replicate showed an aberrant cell frequency that exceeded the normal range. However, this increase was small and was not observed in the replicate culture. As all other cultures (for all treatments) exhibited normal frequencies of numerical aberrations, this increase was not considered of biological importance.

It is concluded that the test material did not induce structural or numerical chromosome aberrations in cultured Chinese hamster ovary (CHO) cells when tested to its limit of cytotoxicity in both the absence and presence of metabolic activation (S-9).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22.03.-30.08.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells were stored as frozen stocks in liquid nitrogen. Full details of the supplier are documented in central records. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37±1 °C. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Metabolic activation:

with and without

Metabolic activation system:

S-9 from male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

3.906 to 250 μg/mL

Vehicle / solvent:

Acetone

- Solubility and stability of the test substance in the solvent/vehicle: The solubility limit in culture medium was in the range of 33.27 to 66.53 μg/mL. The test material was soluble in acetone at concentrations up to at least 212.90 mg/mL.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no reactivity
- Final preparation of a solid: Stock solutions were prepared by formulating the test material under subdued lighting in acetone, with the aid of vortex mixing, to give the maximum required concentration.

Untreated negative controls:

yes

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; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable): 2 x 10 exp 5 cells/mL

DURATION
- Plating for Survival: 7 days
- Expression Period: 7 days
- Plating for Viability: 8 days
- Plating for 6TG Resistance: 13 days

SELECTION AGENT (mutation assays): 6TG

Rationale for test conditions:

According to guideline

Evaluation criteria:

For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
3. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical negative control database
(mean MF +/- 2 standard deviations).
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: limited
- Precipitation: observed at highest tested concentration

Conclusions:

It is concluded that the test material did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of solubility, for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9).

Executive summary:

The information for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD 476.

The test material was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in acetone. A 3 hour treatment incubation period was used for each experiment. In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 15.63 to 500 μg/mL (a precipitating concentration based on data previously generated in a solubility assessment). The highest concentration analysed was 125 μg/mL in the absence and presence of S-9 (limited by the appearance of post-treatment precipitate) which gave 56 % and 174 % relative survival (RS) in the absence and presence of S-9, respectively.

In the Mutation Experiment seven concentrations, ranging from 3.906 to 250 μg/mL, were tested in the absence and presence of S-9. Seven days after treatment, the highest concentration analysed to determine viability and 6TG resistance was 125 μg/mL (limited by the appearance of post treatment precipitate), which gave 48 % and 107 % RS in the absence and presence of S-9, respectively. Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid. No statistically significant increases in MF were observed following treatment with the test material at any concentration analysed in the absence and presence of S-9 and there were no statistically significant linear trends, indicating a clear negative result.

It is concluded that the test material did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of solubility, for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions employed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

OECD 471

Purpose

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.

 

Study Design

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 and TA 1537, and Escherichia coli WP2 uvrA. 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.

 

Results

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 concentrations larger or equal to 1580 µg/plate. Toxicity to the bacteria was not observed.

Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9 -aminoacridine and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as 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.

 

Conclusion

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

 

OECD 473

The test material was tested in an in vitro cytogenetics assay according to OECD 473 using duplicate cultures of Chinese hamster ovary (CHO) cells in two independent experiments. Treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9). The test article was dissolved in sterile ethanol (ethanol) and the highest dose level used, 400 μg/mL, was in excess of the solubility limit in culture medium. In Experiment 1, treatment in the absence and presence of S-9 was for 3 hours followed by a 17-hour recovery period prior to harvest (3+17). The S-9 used was prepared from a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. The test article dose levels for chromosome analysis were selected by evaluating the effect of the test material on relative cell number. Chromosome aberrations were analysed at three dose levels. The highest concentrations chosen for analysis, 27.49 and 83.89 μg/mL induced approximately 58 % and 66 % reduction in cell number and 0 % and 49 % mitotic inhibition (MIH) in the absence and presence of S-9 respectively. In Experiment 2, treatment in the absence of S-9 was continuous for either 20 hours or 44 hours. Treatment in the presence of S-9 was either for 3 hours only followed by a 17-hour recovery period prior to harvest (3+17) or for 3 hours followed by a 41-hour recovery period prior to harvest (3+41). Chromosome aberrations were analysed at two or three dose levels (see overleaf) and the highest concentrations chosen for analysis, 24.57 μg/mL (20+0) or 17.75 μg/mL (44+0) in the absence of S-9 and 81.00 μg/mL (3 +17) or 72.90 μg/mL (3+41) in the presence of S-9, induced approximately 50 %, 60 %, 54 % and 62 % reduction in cell number and 81 %, 29 %, 55 % and 7 % MIH respectively. Appropriate negative (vehicle) control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges. 4-Nitroquinoline 1 -oxide and cyclophosphamide were employed as positive control chemicals in the absence and presence of liver S-9 respectively. Cells receiving these were sampled in each experiment, 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations. Positive controls were included with both treatments in Experiment 1, but only with the 20+0 hour -S-9 and 3+17 +S-9 treatments in Experiment 2.
Treatment of cultures with the test material in the absence and the presence of S-9 (both experiments) resulted in frequencies of cells with structural aberrations which were similar to those observed in concurrent vehicle controls for the majority of concentrations analysed. Two exceptions to this were observed: a single culture at the intermediate concentration analysed (67.11 mg/mL) from the 3+17 hour +S-9 treatment in Experiment 1 and a single culture from the lowest concentration analysed (15.09 mg/mL) from the 44+0 hour –S-9 treatment in Experiment 2 both exhibited numbers of aberrant cells that exceeded historical negative control (normal) values. However, in both instances these increases were not observed in the replicate cultures and were not dose-related. Furthermore, the increase observed in the presence of S-9 in Experiment 1 was not observed at similar concentrations analysed in Experiment 2 (performed under identical treatment conditions). The aberrant cell frequency of all other test material treated cultures fell within normal values. It was therefore considered that the increases observed were spurious and of no biological significance. Normal frequencies of cells with numerical aberrations (within historical negative control (normal) ranges) were observed for the large majority of test material treated cultures. The only exception to this was observed in the 3+17 hour –S-9 treatment at the highest concentration tested (27.49 mg/mL) where a single replicate showed an aberrant cell frequency that exceeded the normal range. However, this increase was small and was not observed in the replicate culture. As all other cultures (for all treatments) exhibited normal frequencies of numerical aberrations, this increase was not considered of biological importance.

It is concluded that the test material did not induce structural or numerical chromosome aberrations in cultured Chinese hamster ovary (CHO) cells when tested to its limit of cytotoxicity in both the absence and presence of metabolic activation (S-9).

 

OECD 476

The information for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD 476.

The test material was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in acetone. A 3 hour treatment incubation period was used for each experiment. In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 15.63 to 500 μg/mL (a precipitating concentration based on data previously generated in a solubility assessment). The highest concentration analysed was 125 μg/mL in the absence and presence of S-9 (limited by the appearance of post-treatment precipitate) which gave 56 % and 174 % relative survival (RS) in the absence and presence of S-9, respectively.

In the Mutation Experiment seven concentrations, ranging from 3.906 to 250 μg/mL, were tested in the absence and presence of S-9. Seven days after treatment, the highest concentration analysed to determine viability and 6TG resistance was 125 μg/mL (limited by the appearance of post treatment precipitate), which gave 48 % and 107 % RS in the absence and presence of S-9, respectively. Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid. No statistically significant increases in MF were observed following treatment with the test material at any concentration analysed in the absence and presence of S-9 and there were no statistically significant linear trends, indicating a clear negative result.

It is concluded that the test material did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of solubility, for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions employed.

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).