Benzyldimethyl(octadecyl)ammonium chloride

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 10, 2017 to November 24, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

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

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

Benzyldimethyl(octadecyl)ammonium chloride

EC Number:

204-527-9

EC Name:

Benzyldimethyl(octadecyl)ammonium chloride

Cas Number:

122-19-0

Molecular formula:

C27H50N.Cl

IUPAC Name:

.

Test material form:

solid

Method

Target gene:

Histidine and tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: see remarks

Remarks:

rfa: deep rough (defective lipopolysaccharide cellcoat); gal: mutation in galactose metabolism; chl: mutation in nitrate reductase; bio defective biotin synthesis; uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: See remarks

Remarks:

rfa: deep rough (defective lipopolysaccharide cellcoat); gal: mutation in galacto se metabolism; chl: mutation in nitrate reductase; bio: defective biotin synthesis; uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)

Metabolic activation:

with and without

Metabolic activation system:

Induction of Liver Enzymes: Male Wistar rats were treated with phenobarbital (PB) and β-naphthoflavone (BNF)

Test concentrations with justification for top dose:

Range finding test: 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the range finding test in tester strains TA100 and TA98 in the absence and presence of metabolic activation.

Initial mutation test:
500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate at Salmonella typhimurium strains with and without metabolic activation (except of without metabolic activation in TA100 and TA1537 strains)

5000, 1581, 500, 158.1, 50 15.81, 5 and 1.581 μg/plate at Escherichia coli WP2 uvrA strain with and without metabolic activation

Complementary initial mutation test: without metabolic activation in TA100 and TA1537 strains 50, 15.81, 5, 1.581, 0.5, 0.1581 and 0.05 μg/plate.

Confirmatory mutation test: 500, 158.1, 50, 15.81, 5, 1.581, 0.5, 0.1581 and 0.05 μg/plate at strains with and without metabolic activation except of without metabolic activation in TA100 and TA1537 strains where the examined concentrations were 50, 15.81, 5, 1.581, 0.5, 0.1581, 0.05 and 0.01581 μg/plate. 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate at E.coli WP2 uvrA strain with metabolic activation.

Complementary confirmatory mutation test: Without metabolic activation in E.coli WP2 uvrA strain 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate.

Vehicle / solvent:

-Vehicle: Water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: See remarks

Details on test system and experimental conditions:

See material and methods

Rationale for test conditions:

See material and methods

Evaluation criteria:

See material and methods

Results and discussion

Test results

Key result

Species / strain:

other: TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Remarks on result:

other: Non genotoxic

Any other information on results incl. tables

Results and discussion:

 Preliminary range finding test (informatory toxicity test): In the preliminary range finding test, the plate incorporation method was used. The preliminary test was performed usingSalmonella typhimuriumTA98 andSalmonella typhimuriumTA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. Each sample (including the controls) was tested in triplicate.

 

Following concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate. In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range where no inhibitory, cytotoxic effect was observed (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system). No precipitate was observed in the preliminary concentration range finding test in both bacterial strains with and without metabolic activation. Inhibitory, cytotoxic effect of the test substance (absent/reduced/sightly reduced background lawn) was detected in the Preliminary Range Finding Test in allSalmonella typhimuriumstrains on the plates at the concentration range of 5000 – 31.6μg/plate without metabolic activation, andat the concentration range of 5000 – 316 or 100μg/plate withmetabolic activation.

 

Based on the results of the range finding test and the solubility findings, the maximum final concentration to be tested in the main experiments was 5000 μg/plate.

  Initial, complementary initial, confirmatory and complementary confirmatory mutation tests (main tests): In the initial mutation test and the complementary initial mutation test, the plate incorporation method was used. In the confirmatory mutation test and the complementary confirmatory mutation test, the pre-incubation method was used. The main tests were carried out using foursalmonella typhimuriumstrains (TA98, TA100, TA1535 and TA1537) and/or theescherichia coliwp2uvrastrain. the main tests were performed in the presence and absence of a metabolic activation system. Each test was performed with appropriate untreated, negative (solvent) and positive controls. In the main tests each sample (including the controls) was tested in triplicate.

 

Based on the results of the range finding test, the test substance concentrations in the Initial Mutation Test were 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581μg/plate atSalmonella typhimuriumstrains with and without metabolic activation (except of without metabolic activation inSalmonella typhimuriumTA100 and TA1537 strains). Moreover, the examined concentrations were 5000, 1581, 500, 158.1, 50 15.81, 5 and 1.581μg/plate atEscherichia coliWP2uvrAstrain with and without metabolic activation.

 

Examined concentrations in the complementary initial mutation test were without metabolic activation inSalmonella typhimuriumTA100 and TA1537 strains 50, 15.81, 5, 1.581, 0.5, 0.1581 and 0.05μg/plate.

  

Examined concentrations in the confirmatory mutation test were 500, 158.1, 50, 15.81, 5, 1.581, 0.5, 0.1581 and 0.05μg/plate atSalmonella typhimuriumstrains with and without metabolic activation except of without metabolic activation inSalmonella typhimuriumTA100 and TA1537 strains where the examined concentrations were 50, 15.81, 5, 1.581, 0.5, 0.1581, 0.05 and 0.01581μg/plate. The examined concentrations were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581μg/plate atEscherichia coliWP2uvrAstrain with metabolic activation.

 

Examined concentrations in the Complementary Confirmatory Mutation Test were without metabolic activation inEscherichia coliWP2uvrAstrain 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581μg/plate.

  

Precipitate/slight precipitate was observed in the confirmatory mutation test in all examinedSalmonellastrains with metabolic activation at the concentrations of 500 and 158.1 μg/plateand inEscherichia coliWP2uvrAstrain with metabolic activation at the concentrations of 5000 and1581 μg/plate. The precipitation did not adversely affect the colony counting.

 

Inhibitory, cytotoxic effect of the test substance (absent/reduced/slightly reduced background lawn development) was observed in the initial mutation test and the complementary initial mutation test in allSalmonella typhimuriumstrains on the plates at 500 and 158.1μg/plate concentrations with metabolic activation and 500, 158.1, 50μg/plate concentrations without metabolic activation except of without

metabolic activation inSalmonella typhimuriumTA100 and TA1537 strains where the cytotoxic effect were observed at 50 and 15.81μg/plate concentrations. The similar cytotoxic effect was observed inEscherichia coliWP2uvrAstrain with and without metabolic activation at 5000 and 1581μg/plate concentrations.

 

In the confirmatory mutation test and the complementary confirmatory mutation Test absent/reduced/slightly reduced background lawn was observed in allSalmonella typhimuriumstrains on the plates at 500, 158.1 and 50μg/plate concentrations with metabolic activation and at 500, 158.1, 50 and/or 15.81μg/plate concentrations without metabolic activation except of without metabolic activation inSalmonella typhimuriumTA100 and TA1537 strains where reduced/slightly reduced background lawn was observed at 50, 15.81 and 5μg/plate concentrations. The similar effect was detected inEscherichia coliWP2uvrAstrain without metabolic activation at 500, 158.1 and 50μg/plate concentrationsand with metabolic activation at 5000, 1581 and 500μg/plate concentrations.

 

In the main tests, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible doserelated trends and there was no indication of any treatment-related effect.

 

In the initial mutation test and the complementary initial mutation test (plate incorporation method), the highest revertant rate was observed inSalmonella typhimuriumTA98 strain at 0.1581 μg/plate concentration withmetabolic activation (the observed mutation factor value was: MF: 1.46). However, there was no dose response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.

 

 In the confirmatory mutation test complementary confirmatory mutation test (preincubation method), the highest revertant rate was observed inSalmonella typhimuriumTA1535 bacterial strain at 0.1581μg/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.29). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range. Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test. Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological

variability of the test system.

 

Validity of the tests

Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. At least five analysable concentrations were presented in all strains with and without metabolic activation.

  

The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was determined to be not-mutagenic in the bacterial reverse mutation assay, with and without metabolic activation.

Executive summary:

A study was conducted to determine the in vitro genotoxic potential of the test substance, C18 ADBAC (active: 96.2%), using Ames test, according to OECD Guideline 471and EU Method B.13/14, in compliance with GLP. The experiments were carried out using strains of Salmonella typhimurium (TA98, A100, TA1535 and TA1537) and a strain of Escherichia coli WP2uvrA in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone induced rats. The study included a (a) preliminary compatibility test, (b) a preliminary range finding test (informatory toxicity test), (c) an initial mutation test (plate incorporation method), (d) a complementary initial mutation test (plate incorporation method), which was repeated with selective strains due to excessive cytotoxicity), (e) a confirmatory mutation test (pre-incubation method) and a complementary confirmatory mutation test (pre-incubation method), which was repeated with selective strains due to excessive cytotoxicity. Based on the results of the compatibility test, test concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in a range finding test using TA100 and TA98 tester strains, in the absence (-S9) and presence (+S9) of metabolic activation. Based on the results of the range finding test, the selected main test concentrations included: 0.01581, 0.05, 0.1581, 0.5, 1.581, 5, 15.81, 50, 158.1, 500, 1581, 5000 μg/plate. In the initial and complementary initial mutation tests, S. typhimurium and E.coli strains were treated with test concentrations ranging from 0.1581 to 500 μg/plate and 1.581 to 5000 μg/plate, with and without S9. Due to excessive cytotoxicity, the S. typhimurium TA100 and TA1537 strains were treated with modified concentrations ranging from 0.05 to 50 μg/plate, without S9. In the confirmatory and complementary confirmatory mutation tests, S. typhimurium and E.coli strains were treated with test concentrations ranging from 0.05 to 500 μg/plate and 1.581 to 5000 μg/plate, with and without S9. Due to excessive cytotoxicity, the S. typhimurium TA100 and TA1537 strains were treated with modified concentrations ranging from 0.01581 to 50 μg/plate, without S9.

Precipitate or slight precipitate was observed in the confirmatory mutation test in all examined Salmonella strains with S9 at the concentrations of 500 and 158.1μg/plate and in E.coli WP2uvrA strain with S9 at the concentrations of 5000 and1581 μg/plate. The precipitation did not adversely affect the colony counting. Inhibitory, cytotoxic effect of the test substance was observed in the initial mutation test and the complementary initial mutation test in all S. typhimurium strains on the plates at 500 and 158.1 μg/plate concentrations with S9 and 500, 158.1, 50 μg/plate concentrations without S9, except without S9 in TA100 and TA1537 strains, where the cytotoxic effect were observed at 50 and 15.81 μg/plate concentrations. Similar cytotoxic effect was observed in E.coli WP2uvrA strain with and without S9 at 5000 and 1581 μg/plate concentrations. In the confirmatory mutation test and the complementary confirmatory mutation test absent/reduced/slightly reduced background lawn was observed in all S. typhimurium strains on the plates at 500, 158.1 and 50 μg/plate concentrations with S9 and at 500, 158.1, 50 and/or 15.81 μg/plate concentrations without S9 except for without S9 in TA100 and TA1537 strains, where reduced/slightly reduced background lawn was observed at 50, 15.81 and 5 μg/plate concentrations. The similar effect was detected in E.coli WP2uvrA strain without S9 at 500, 158.1 and 50 μg/plate concentrations and with S9 activation at 5000, 1581 and 500 μg/plate concentrations. In the main tests, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose related trends and no indication of any treatment-related effect. The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range. The reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests and the examined concentration range was considered to be adequate. Therefore, the study was considered to be valid. Under the study conditions, the test substance was determined to be not-mutagenic in the bacterial reverse mutation assay, with and without metabolic activation (Kovács, 2018).