Benzyldimethyl(octadecyl)ammonium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of the Ames test with the test substance as well as two in vitro assays with the read across substance, the test substance, C18 ADBAC, is considered to be not genotoxic

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:

From November 10, 2017 to November 24, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

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:

Histidine and tryptophan

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 / 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

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

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

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.

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).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Refer to the section 13 of IUCLID dataset for details on the read across justification. The algae study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

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)

Metabolic activation:

with and without

Metabolic activation system:

S9 from rats treated with Aroclor 1254 (S-9 mix)

Test concentrations with justification for top dose:

Based on preliminary cytotoxicity test: 0, 0.36, 1.09, 3.27 and 9.80 µg/mL (without S9); 0, 6.13, 12.25, 24.5, 49.0 and 98.0 µg/mL (with metabolic activation of S9 from rats treated with Aroclor 1254).

Details on test system and experimental conditions:

Exposure times were 21h for the experiments without metabolic activation, and 3h for the experiments with metabolic activation, with harvesting times of 12 and 21h.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: >= 15.68 µg/mL (- S9); >= 78.4 µg/mL (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- A preliminary toxicity test, showed that in the absence of S9-mix, the substance was toxic in concentrations of 15.68 µg/mL and above; in the presence of S9-mix, the substance was very toxic at 78.4 µg/mL and above at both harvesting times.
- The test substance did not induce a statistically significant increase in the number of cells with structural chromosome aberrations (breaks, exchanges and multiple aberrations) at any of the concentrations used, either in the absence or in the presence of the S-9 mix.

Remarks on result:

other: non-genotoxic

Conclusions:

Based on the results of the read across study, the test substance is not considered to be clastogenic in Chinese hamster ovary cells with or without metabolic activation.

Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the read across substance, C12-16 ADBAC (active: 99.24%) using Chinese hamster ovary cells, according to OECD Guideline 473 (chromosome aberration test), in compliance with GLP. Based on preliminary cytotoxicity test, 0, 0.36, 1.09, 3.27 and 9.80 µg/mL test concentrations (without S9) as well as 0, 6.13, 12.25, 24.5, 49.0 and 98.0 µg/mL (with metabolic activation of S9 from rats treated with Aroclor 1254) test concentrations were selected. The exposure times were 21 h for the experiments without metabolic activation, and 3 h for the experiments with metabolic activation, with harvesting times of 12 and 21 h. In the preliminary toxicity test, in the absence of S9-mix, the substance was toxic at a concentration of 15.68 µg/mL and above; and in the presence of S9-mix, the substance was very toxic at 78.4 µg/mL and above at both harvesting times. The read across substance did not induce a statistically significant increase in the number of cells with structural chromosome aberrations (breaks, exchanges and multiple aberrations) at any of the concentrations used, either in the absence or in the presence of the S-9 mix. Under the conditions of the study, the read across substance was not considered to be clastogenic in the chromosomal aberration assay, with or without metabolic activation (Willmer, 1986). Based on the results of the read across study, similar non-clastogenic behaviour is considered for the test substance, C18 ADBAC in the chromosomal aberration assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Justification for type of information:

Refer to the section 13 of IUCLID dataset for details on the read across justification. The algae study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Based on a preliminary cytotoxicity test:
0, 5, 10, 12.5, 15, 17.5 and 20 nL/mL (without metabolic activation) and
0, 15, 30, 45, 60, 75 and 90 nL/mL (with metabolic activation)

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

- The point mutation test was carried out in duplicate both in the presence and the absence of a metabolic activation system.
- Exposure duration of 5h.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: > 17.5 nL/mL (- S9), >= 70 nL/mL (+ S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Concentration-dependent increases in the number of mutants at the HGPRT locus were not observed either with or without metabolic activation. Exposure to the highest concentrations tested resulted in significant (initial) toxicity for the CHO cells; this indicates that the test was carried out with appropriate concentrations of the test substance. Exposure to either EMS or DMN gave the expected increases in the mutation frequency. 
The toxicity of the test substance for CHO cells was clearly reduced in the presence of metabolic activation.

Conclusions:

Based on the results of the study, test substance is not considered to show mutagenic activity at the HGPRT locus in CHO cells with and without metabolic activation

Executive summary:

A study was conducted to determine the in vitro genotoxic potential of the read across substance, C12 -16 ADBAC, at HGPRT locus in Chinese hamster ovary (CHO) cells, according to OECD Guideline 476 (cell gene mutation test), in compliance with GLP. Based on a preliminary toxicity test, 0, 5, 10, 12.5, 15, 17.5 and 20 nL/mL (without metabolic activation) and 0, 15, 30, 45, 60, 75 and 90 nL/mL (with metabolic activation) were selected for the experiment. The entire experiment was repeated to confirm the results of the first experiment. Five hour exposure was used both with and without S9-mix. The test substance did not induce dose-related increase in the mutant frequency at the HGPRT locus at any dose level, either with or without metabolic activation. Exposure to the highest concentrations tested resulted in significant (initial) toxicity for the CHO cells; this indicates that the test was carried out with appropriate concentrations of the test substance. Exposure to positive control substances gave the expected increases in the mutation frequency. The toxicity of the test substance for CHO cells was clearly reduced in the presence of metabolic activation. Under the study conditions, the read across substance was not considered to show mutagenic activity at the HGPRT locus in CHO cells with and without metabolic activation (Davis, 1986). Based on the results of the read across study, the test substance, C18 ADBAC, is considered to be non-mutagenic in the HGPRT assay, with and without metabolic activition.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Study 1: 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).

Study 2: A study was conducted to determine the in vitro genetic toxicity of the read across substance, C12-16 ADBAC (active: 99.24%) using Chinese hamster ovary cells, according to OECD Guideline 473 (chromosome aberration test), in compliance with GLP. Based on preliminary cytotoxicity test, 0, 0.36, 1.09, 3.27 and 9.80 µg/mL test concentrations (without S9) as well as 0, 6.13, 12.25, 24.5, 49.0 and 98.0 µg/mL (with metabolic activation of S9 from rats treated with Aroclor 1254) test concentrations were selected. The exposure times were 21 h for the experiments without metabolic activation, and 3 h for the experiments with metabolic activation, with harvesting times of 12 and 21 h. In the preliminary toxicity test, in the absence of S9-mix, the substance was toxic at a concentration of 15.68 µg/mL and above; and in the presence of S9-mix, the substance was very toxic at 78.4 µg/mL and above at both harvesting times. The read across substance did not induce a statistically significant increase in the number of cells with structural chromosome aberrations (breaks, exchanges and multiple aberrations) at any of the concentrations used, either in the absence or in the presence of the S-9 mix. Under the conditions of the study, the read across substance was not considered to be clastogenic in the chromosomal aberration assay, with or without metabolic activation (Willmer, 1986). Based on the results of the read across study, similar non-clastogenic behaviour is considered for the test substance, C18 ADBAC in the chromosomal aberration assay.

Study 3: A study was conducted to determine the in vitro genotoxic potential of the read across substance, C12 -16 ADBAC, at HGPRT locus in Chinese hamster ovary (CHO) cells, according to OECD Guideline 476 (cell gene mutation test), in compliance with GLP. Based on a preliminary toxicity test, 0, 5, 10, 12.5, 15, 17.5 and 20 nL/mL (without metabolic activation) and 0, 15, 30, 45, 60, 75 and 90 nL/mL (with metabolic activation) were selected for the experiment. The entire experiment was repeated to confirm the results of the first experiment. Five hour exposure was used both with and without S9-mix. The test substance did not induce dose-related increase in the mutant frequency at the HGPRT locus at any dose level, either with or without metabolic activation. Exposure to the highest concentrations tested resulted in significant (initial) toxicity for the CHO cells; this indicates that the test was carried out with appropriate concentrations of the test substance. Exposure to positive control substances gave the expected increases in the mutation frequency. The toxicity of the test substance for CHO cells was clearly reduced in the presence of metabolic activation. Under the study conditions, the read across substance was not considered to show mutagenic activity at the HGPRT locus in CHO cells with and without metabolic activation (Davis, 1986). Based on the results of the read across study, the test substance, C18 ADBAC, is considered to be non-mutagenic in the HGPRT assay, with and without metabolic activition.

Justification for classification or non-classification

Based on the results of the Ames test with the test substance as well as two in vitro assays with the read across substance, the test substance, C18 ADBAC, is concluded not to warrant classification for genotoxicity according to EU CLP criteria (Regulation 1272/2008/EC).