1,4-phenylenebis(nitrilo-2,2-dimethylprop-1-yl-3-ylidene) didodecanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: The test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used.

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:

2015-04-16 and 2015-06-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

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:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use (June 2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Trinova Biochem GmbH (Rathenau Str. 2 (earlier: Kerkrader Str. 10));
D-35394 Giessen, Germany

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver

Test concentrations with justification for top dose:

5000, 1600, 500, 160, 50, 16 and 5 μg/plate (inital and cofirmatory mutation test)
Exception: TA 1537 (+ S9 mix): 1600, 500, 160, 50, 16, 5 and 1.6 μg/plate (confirmatory mutation test)

Vehicle / solvent:

acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine (NPD)

Remarks:

Strain: TA 98, wihtout metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Strains: TA 100 and TA 1535, without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Strain: TA 1537, without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Strain: E.coli WP2 uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

All strains, with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min (preincubation method)
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: determination of the number of revertant colonies

Rationale for test conditions:

According to guidelines

Evaluation criteria:

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

Criteria for a Negative Response:
A test article is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the performed experiments the test item solution precipitated in a form of drops (“microdrops”) on the plates; in all examined strains at the concentrations of 5000 and 1600 μg/plate, without and with addition of metabolic activation (±S9 Mix) following the plate incorporation procedure (Initial Mutation Test) and at the concentration level of 5000 μg/plate, without metabolic activation (-S9 Mix) following the pre-incubation procedure (Confirmatory Mutation Test).

RANGE-FINDING/SCREENING STUDIES: Based on the pre-experiment study, the following concentrations were examined in the initial and confirmatory mutation test: 5000, 1600, 500, 160, 50, 16 and 5 µg/plate. In the confirmatory mutation test slight modification of the concentrations were done in Salmonella typhimurium TA 1537 (+ S9 mix), the following concentrations were examined: 1600, 500, 160, 50, 16, 5 and 1.6 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes; The spontaneous revertant colony numbers of the acetone vehicle control plates showed mostly characteristic mean numbers agreed with the actual historical control data ranges in the main experiments; however the spontaneous revertant colony numbers of the acetone vehicle control plates were slightly than the chracteristic mean numbers agreed with the actual historical data range in the initial mutation test in S. typhimurium TA100 (- S9 mix). The slightly higher counts were evaluated as acceptable without any influence on the final conclusion of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Signs of cytotoxicity has been observed in all tested strains with and/or without metabolic activation. The 500 µg/plate was found to be the lowest cytotoxic concentration, observed in the case of Salmonella typhimurium strains, in the presence of exogenous metabolic activation (+S9 mix).

For summary of results see attached background material.

Conclusions:

The reported data of this mutagenicity assay shows, that the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

To investigate the mutagenic potential of the test item, a Bacterial Reverse Mutation Assay (using Salmonella typhimurium TA 98, TA 1537, TA 1535, TA 100 and Escherichia coli WP2 uvrA) was conducted with and without metabolic activation system (S9 mix). The test item was dissolved (mixed with) in acetone. In the Initial (experiment I, plate incorporation test) and Confirmatory Mutation (experiment II, preincubation test) Tests the following concentrations were examined: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate. In the Confirmatory Mutation Test slight modification of the concentrations was done in Salmonella typhimurium TA1537, in presence of metabolic activation (+S9 Mix), the originally planned concentration levels were lowered and the following concentrations were examined: 5000; 1600, 500, 160, 50, 16, 5 and 1.6 μg/plate. The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values mostly within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. In the performed experiments inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was observed in all examined bacterial strains. The 500 μg/plate was found to be the lowest cytotoxic concentration, observed in the case of Salmonella typhimurium strains, in the presence of exogenous metabolic activation (+S9 Mix). In conclusion, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

To investigate the mutagenic potential of the test item, a Bacterial Reverse Mutation Assay (using Salmonella typhimurium TA 98, TA 1537, TA 1535, TA 100 and Escherichia coli WP2 uvrA) was conducted with and without metabolic activation system (S9 mix). The test item was dissolved (mixed with) in acetone. In the Initial (experiment I, plate incorporation test) and Confirmatory Mutation (experiment II, preincubation test) Tests the following concentrations were examined: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate. In the Confirmatory Mutation Test slight modification of the concentrations was done in Salmonella typhimurium TA1537, in presence of metabolic activation (+S9 Mix), the originally planned concentration levels were lowered and the following concentrations were examined: 5000; 1600, 500, 160, 50, 16, 5 and 1.6 μg/plate. The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values mostly within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. In the performed experiments inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was observed in all examined bacterial strains. The 500 μg/plate was found to be the lowest cytotoxic concentration, observed in the case of Salmonella typhimurium strains, in the presence of exogenous metabolic activation (+S9 Mix). In conclusion, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used.

Justification for classification or non-classification

Based on available data of the bacterial reverse mutation test, the test item is not classified and labelled as mutagenic according to Regulation (EC) No 1272/2008 (CLP).