Quaternary ammonium compounds, C12-14-alkyl[(ethylphenyl)methyl]dimethyl, chlorides

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 21, 2017 to December 18, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

impurity 1

Test material form:

not specified

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Mitochondrial supernatant (S9) prepared from livers of phenobarbital/β-naphthoflavone-induced rats

Test concentrations with justification for top dose:

Preliminary test: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Experiment 1: 0.0316, 0.100, 0.316, 1.0, 3.16, 10.0, 31.6 and 100 µg/plate
Experiment 2: 0.050, 0.158, 0.50, 1.58, 5.00, 15.8, 50.0 and 100 µg/plate

Vehicle / solvent:

Purified water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene and 4-nitro-o-phenylene-diamine

Details on test system and experimental conditions:

Preparation of the Test Substance
The test substance was dissolved in A. dest. and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity. A correction factor of 2.0 was applied to consider the purity of the test substance.

Controls
Solvent / negative as well as positive controls were included in each experiment. Strain specific positive controls were included in the assay, which demonstrated the effective performance of the test.

Negative/Solvent Controls
Negative/solvent controls (A. dest., Eurofins Munich, Lot No. 171107, 171118, 171004) were treated in the same way as all dose groups.

Positive Controls
Without metabolic activation
Tester Strains: S. typhimurium: TA 100, TA 1535
Name: NaN3; sodium azide
CAS No.: 26628-22-8
Supplier: Sigma
Batch No.: STBF8665V
Dissolved in: A. dest.
Concentration: 10 µg/plate
 
Tester Strains: S. typhimurium: TA 98, TA 1537
Name: 4-NOPD; 4-nitro-o-phenylene-diamine
CAS No.: 99-56-9
Supplier: Fluka
Batch No.: MKBQ2637V
Dissolved in: DMSO
Concentrations: 10 µg/plate for TA 98, 40 µg/plate for TA 1537

Tester Strain: S. typhimurium: TA 102
Name: MMS; methylmethanesulfonate
CAS No.: 66-27-3
Supplier: Sigma
Batch No.: MKBX5165V
Dissolved in: A. dest.
Concentration: 1 µL/plate

With metabolic activation
Tester Strains: S. typhimurium: TA 98, TA 100, TA 1535, TA 1537 and TA 102
Name: 2-AA; 2-aminoanthracene
CAS No.: 613-13-8
Supplier: Aldrich
Batch No.: STBD3302V
Dissolved in: DMSO
Concentrations: 2.5 µg/plate; 10 µg/plate for TA 102
The stability of the positive control substances in solution is unknown but a mutagenic response in the expected range is sufficient evidence of biological stability.

Test System: Bacteria
Five strains of S. typhimurium with the following characteristics were used:
TA 98: his D 3052; rfa-; uvrB-; R-factor: frame shift mutations
TA 100: his G 46; rfa-; uvrB-; R-factor: base-pair substitutions
TA 1535: his G 46; rfa-; uvrB-: base-pair substitutions
TA 1537: his C 3076; rfa-; uvrB-: frame shift mutations
TA 102: his G 428 (pAQ1); rfa-; R-factor: base-pair substitutions

Tester strains TA 98, TA 1535 and TA 102 were obtained from MOLTOX, INC., NC 28607, USA. Tester strains TA 100 and TA 1537 were obtained from Xenometrix AG, Switzerland. They were stored as stock cultures in ampoules with nutrient broth (OXOID) supplemented with DMSO (approx. 8% v/v) over liquid nitrogen. All Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. They contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain of the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene coding for a protein of the DNA nucleotide excision repair system resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chl) and biotin (bio) genes (bacteria require biotin for growth). The tester strains TA 98, TA 100 and TA 102 contain the R-factor plasmid, pkM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non R-factor parent strains. pkM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system which is normally present in these organisms. The properties of the S. typhimurium strains with regard to membrane permeability, ampicillin- and tetracycline-resistance as well as normal spontaneous mutation rates are checked regularly according to Ames et al. In this way it is ensured that the experimental conditions set up by Ames are fulfilled.

S9 Homogenate
The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.
The following quality control determinations are performed:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL and stored at ≤75 °C. The protein concentration in the S9 preparation (Lot: 150917, 020617) was 34.0 and 34.2 mg/mL, respectively.

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test substance is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. A test substance producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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

Any other information on results incl. tables

Results

No precipitation of the test substance was observed in any tester strain used in experiment I and II (with and without metabolic activation). Toxic effects of the test substance were noted in all tester strains evaluated in experiment I and II. In experiment I toxic effects of the test substance were observed in tester strain TA 98 at concentrations of 10.0 µg/plate and higher (without metabolic activation) and at concentrations of 31.6 µg/plate (with metabolic activation). In tester strain TA 100 toxic effects of the test substance were noted at concentrations of 31.6 µg/plate and higher (without metabolic activation) and at concentrations of 100 µg/plate (with metabolic activation). In tester strain TA 1535 toxic effects of the test substance were noted at concentrations of 10.0 µg/plate and higher (with and without metabolic activation). In tester strain TA 1537 toxic effects of the test substance were noted at concentrations of 31.6 µg/plate and higher (with and without metabolic activation). In tester strain TA 102 toxic effects of the test substance were observed at concentrations of 10.0 µg/plate and higher (without metabolic activation) and at concentrations of 100 µg/plate and higher (with metabolic activation). In experiment II toxic effects of the test substance were noted in tester strains TA 98 and TA 1535 at concentrations of 15.8 µg/plate and higher (without metabolic activation) and at concentrations of 50.0 µg/plate and higher (with metabolic activation). In tester strains TA 100, TA 1537 and TA 102 toxic effects of the test substance were noted at concentrations of 15.8 µg/plate and higher (without metabolic activation) and at a concentration of 100 µg/plate (with metabolic activation). No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Quaternary ammonium compounds, C12-14-alkyl[(ethylphenyl) methyl]dimethyl, chlorides at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. All criteria of validity were met.

For detailed tables, kindly refer the attached background material section.

Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Quaternary ammonium compounds, C12-14-alkyl[(ethylphenyl) methyl]dimethyl, chlorides did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Quaternary ammonium compounds, C12-14-alkyl[(ethylphenyl)methyl]dimethyl, chlorides is considered to be non-mutagenic in this bacterial reverse mutation assay.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was determined to be non-mutagenic in the Ames test, with and without metabolic activation.

Executive summary:

An in vitro study was conducted to determine the genotoxic potential of test substance, C12-14 ADEBAC (active: 51.13%), using Ames test, according to OECD Guideline 471 and EU Method B13/14, in compliance with GLP. In two independent experiments, Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 were treated with suspensions of the test substance using the Ames plate incorporation at up to eight dose levels (0.0316 to 100 µg/plate), in triplicate, both with and without metabolic activation (liver S9 in standard co-factors). The doses for experiement 1 and experiement 2 were selected based on preliminary study results. The dose range for experiment 1 and experiment 2 were ranged from 0.0316 to 100 µg/plate (0.0316, 0.100, 0.316, 1.0, 3.16, 10.0, 31.6 and 100 µg/plate) and from 0.050 to 100 µg/plate (0.050, 0.158, 0.50, 1.58, 5.00, 15.8, 50.0 and 100 µg/plate), respectively. No precipitation of the test substance was observed in any tester strain used in experiment I and II (with and without metabolic activation). In experiment I toxic effects of the test substance were observed at concentrations of 10.0 µg/plate and higher (with and without metabolic activation), depending on the particular tester strain. In experiment II toxic effects of the test substance were noted at concentrations of 15.8 µg/plate and higher (without metabolic activation) and at concentrations of 50.0 µg/plate and higher (with metabolic activation), depending on the particular tester strain. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with test substance at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. All criteria of validity were met. Under the study conditions, the test substance was determined to be non-mutagenic in the Ames test, with and without metabolic activation (Schreib, 2017).