4-chloromethyl-4''-ethyl-2'-fluoro-[1,1':4',1''-terphenyl]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test (reference 7.6.1-1).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-11-08 to 2011-11-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

07-1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

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

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:

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:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: mutations in the tryptophan operon

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: from homogenized livers of male Wistar, HSdCpb:Wu rats, aged 6-8 weeks, with single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) dissolved in Miglyol 812 oil
- method of preparation of S9 mix: Please refer to “Any other information on materials and methods”.
- concentration or volume of S9 mix and S9 in the final culture medium: 10 % and 30 % S9 in the S9 mix are used in the 1st and 2nd test series, respectively.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): S9-batch was tested for its metabolic activity by the use of specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene was thus determined once for the S9-batch. Clear increases in the number of revertants for S. typhimurium TA 98, TA 100, and TA 1537 with all positive controls and for TA 1535 with 2-aminoanthracene were used as an acceptance criterion for the S9-batch. In this study 2-aminoanthracene, and benzo[a]pyrene for TA 102, were used as the concurrent positive controls for the different strains in the presence of S9 mix.

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:
1st series: 5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate
2nd series: 5, 15.8, 28.1, 50, 88.9 and 158 µg/plate

5000 µg/plate was chosen as the appropriate maximum test material concentration.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

cumene hydroperoxide

other: daunomycin

Remarks:

without S9 mix

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 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): 3 plates for test item and positive control, 6 for negative control
- Number of independent experiments: 2

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

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2 to 3 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; reduction in number of spontaneous revertants

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The assessment of test material-induced effects was 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 criteria, based upon the historical controls of the laboratory and statistical considerations, were established as shown in table 2 (see “Any other information on materials and methods”).

Rationale for test conditions:

Test conditions in line with respective test guidelines were used.

Evaluation criteria:

A test material was 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 was defined as mutagenic in this assay if
- a dose-related (over at least two test material concentrations) increase in the number of revertants was induced, the maximal effect was a "clear increase", and the effects were reproduced at similar concentration levels in the same test system;
- "clear increases" occur at least at one test material concentration, higher concentrations showed strong precipitation or cytotoxicity, and the effects were 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 and the results of each series be discussed case by case.

Statistics:

not specified

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 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 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 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:

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:

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:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not specified
- Data on osmolality: not specified
- Possibility of evaporation from medium: not specified
- Water solubility: Please refer to respective IUCLID section.
- Precipitation and time of the determination: Precipitation of the test material on the agar plates occurred at concentrations ≥158 µg/plate and was observed at plate counting.
- Definition of acceptable cells for analysis: not applicable
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: Doses in the 2nd experiment were adjusted based observations from 1st experiment.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Please refer to attached document under "Overall remarks, attachments".

Ames test:
- Signs of toxicity: No toxicity was observed.
- Individual plate counts: Please refer to attached document under "Overall remarks, attachments".
- Mean number of revertant colonies per plate and standard deviation: Please refer to attached document under "Overall remarks, attachments".

HISTORICAL CONTROL DATA
Not specified

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:

The investigations for the mutagenic potential of the test item were performed according to OECD TG 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. In the two series with S9 mix, 10 % S9 in the S9 mix were used in the 1st and 30 % in the 2nd series, respectively. The test item was dissolved in DMSO and tested at concentrations ranging from 5 - 5000 μg/plate. Precipitation of the test material on the agar plates occurred at concentrations of >= 158 μg/plate. Toxicity to the bacteria was not observed. Each treatment with 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. With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The investigations for the mutagenic potential of the test item were performed according to OECD TG 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. In the two series with S9 mix, 10 % S9 in the S9 mix were used in the 1st and 30 % in the 2nd series, respectively. The test item was dissolved in DMSO and tested at concentrations ranging from 5 - 5000 μg/plate. Precipitation of the test material on the agar plates occurred at concentrations of >= 158 μg/plate. Toxicity to the bacteria was not observed. Each treatment with 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. With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.

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 the test item does not require classification for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP).