Quaternary ammonium compounds, coco alkyltrimethyl, chlorides

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of the three in vitro studies, the test substance is considered to have no genotoxicity potential.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Ames test

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 18 April, 1989 to 08 May, 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

S. Typhimurium TA102 and/or E.coli WP2 strains not included; conducted according to old version of Guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

the version of the OECD guideline should be specified: OECD 471 (1983).

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

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 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

1, 3, 10, 32 and 100 µg/plate for both with and without S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Tested with TA 1535, TA 100 in the absence of S-9 mix

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Tested for TA 1535 in the presence and absence of S9 mix

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Tested for TA 1537 in the absence of S9-mix

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Tested for TA 98 in the absence of S9-mix

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Tested for TA 1537, TA 100 and TA 98 in the presence and absence of S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (pour-plate method)

DURATION
- Incubation period: 48 h at approximately 37°C

NUMBER OF REPLICATIONS: Three

DETERMINATION OF CYTOTOXICITY
- Method: Number of revertant colonies were counted, either manually or with a Biotran II automatic colony counter. Total colonies on nutrient plates were counted in the same way. Growth of the background lawn of non-revertant cells on minimal plates were verified.

Evaluation criteria:

The number of the revertants per plate of the tester strain is compared to the control.

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

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

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

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

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test: Yes

Remarks on result:

other: not mutagenic

Conclusions:

Under the study conditions, the test substance is not considered to be mutagenic in the presence and absence of exogenous metabolic activation.

Executive summary:

A study was conducted to determine the mutagenic potential of test substance, Coco TMAC (33% active in water), according to OECD 471 and EPA OPPTS 870.5265 Guidelines, in compliance with GLP. Using pour-plate assays, the test substance was examined for mutagenic activity in four strains of Salmonella typhimurium: TA 100, TA 1535, TA 1537 and TA 98 in the absence and in the presence of S9-mix. The test concentrations included 0, 1, 3, 10, 32 and 100 µg/plate (i.e., equivalent to 0, 0.33, 0.99, 3.3, 10.56 and 33 µg/plate). No increase in reversion to prototrophy was obtained with any of the bacterial strain at any concentration level either in the presence or absence of S9-mix. Further, inhibition of growth, observed as thinning of the background lawn of non-revertant cells, occurred in all strains following exposure to the test substance at 100 µg/plate.Appropriate positive control chemicals (with S-9 mix where required) induced marked increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S-9 mix. Under the study conditions, the test substance was not mutagenic with and without metabolic activation (May, 1989).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Mouse lymphoma assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 15 October, 2001 to 05 March, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission directive 2002/32/EC and U.K Environmental Mutagen Society

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

For Experiment 1: 0.625, 1.25, 2.5, 5, 10 and 20 µg/mL for both with and without S9-mix
For Experiment 2: 0.313, 0.625, 0.938, 1.25, 2.5 and 5 µg/mL (without S9 mix) and 2.5, 5, 10, 15, 20 and 30 µg/mL (with S9-mix)

Vehicle / solvent:

RPMI 1640 without serum

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

in the absence of S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

in the presence of S9-mix

Details on test system and experimental conditions:

After a preliminary toxicity test, 0.625, 1.25, 2.5, 5, 10 and 20 µg/L was selected for the first experiment. The entire experiment was repeated to confirm the results of the first experiment. 3 h exposure were used both with and without S9 mix while for the second experiment, the exposure time was increased to 24 h. For the second experiment, the dose range was 2.5 to 30 µg/L with S9 mix and 0.313 to 5 µg/L without S9 mix.

Evaluation criteria:

For a test substance to give a significant result then two or more of the following criteria should be met:
a) A greater than three-fold increase in the mutant frequency/survivor over the negative control value.
b) A dose-related increase in the mutant frequency per survivor.
c) An increase in the absolute number of mutants.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

RANGE-FINDING/SCREENING STUDIES: Yes

Remarks on result:

other: not mutagenic

There was evidence of toxicity with the test substance in both the presence and absence of S9-mix as indicated by relative suspension growth (RSG), this was confirmed by the decrease in relative total growth (RTG). There was no evidence of a reduction in Day 2 viability, therefore indicating that no residual toxicity occurred, in either the presence or absence of S9-mix. Optimum level of toxicity was achieved, in both the presence and absence of S9-mix. The toxicity observed at 30 µg/mL in the presence of metabolic activation exceeded the upper acceptable limit of 90%; therefore it was excluded from the statistical analysis.

Conclusions:

Under the study conditions, the test substance was not found to show mutagenic activity in mouse lymphoma assay.

Executive summary:

A study was conducted to determine the potential of the test substance, Coco TMAC (35.5% active in water), to induce mutations at the TK (thymidine kinase) locus in L5178Y mouse lymphoma cells. Study was performed accoding to OECD 476 Guideline, EU Method B.17 and Commission directive 2002/32/EC and U.K Environmental Mutagen Society, in compliance with GLP. After a preliminary toxicity test, 0.625, 1.25, 2.5, 5, 10 and 20 µg/L was selected as the test concentrations for the first experiment. The experiment was repeated to confirm the results of the first experiment. Three hour exposures were used both with and without S9-mix while for the second experiment, the exposure time was increased to 24 h. For the second experiment, the concentration range was 2.5 to 30 µg/L with S9-mix and 0.313 to 5 µg/L without S9-mix. The test substance did not induce a statistically significant or concentration-related increase in the mutant frequency at any concentration level, either with or without metabolic activation. Adequate levels of toxicity were achieved in all exposure groups. Under the study conditions, the test substance was not found to show mutagenic activity in mouse lymphoma assay (Nolan, 2002).

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

chromosome aberration assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 18 April, 1989 to 09 May, 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Cultured peripheral human lymphocytes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

- without S9-mix: 0.4, 2 and 10 µg/L
- With S9-mix: 2, 10 and 50 µg/L

Vehicle / solvent:

Sterile distilled water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: chlorambucil

Remarks:

without S9-mix

Details on test system and experimental conditions:

Study Type: Other: Chromosomal aberration test
Organism/cell type: Cultured peripheral human lymphocytes
Metabolic activation system: S9 mix
Positive control : Chlorambucil (without S9 mix), cyclophosphamide (with S9-mix)

Statistics:

Data from each treatment was compared with the respective solvent control group using Fisher Exact Probability test. A one-sided test was applied and p <0.05 was used as the lowest level of significance.

Key result

Species / strain:

lymphocytes: cultured human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

An increased incidences in polyploidy would normally be associated with spindle dysfunction; such a event would also be expected to increase the incidence of aneuploidy and if this occurred in vivo, micronuclei would be produced in the target cell population. However, a mouse bone marrow micronuclei test, performed elsewhere has no induction following oral administration of the test substance.

Remarks on result:

other: not clastogenic

Conclusions:

Under the study conditions, the test substance was not found to be clastrogenic in human lymphocytes.

Executive summary:

A study was conducted to determine the clastogenic potential of the test substance, Coco TMAC (33% active in water), in cultured human lymphocytes according to OECD Guideline 473, in compliance with GLP. After a concentration range finding test, two independent tests were performed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (S9-mix). With S9-mix cells were exposed for 24 hours. The test substance produced a reduction in mitotic activity (i.e., approximately 48%) at 10 µg/L in the absence of S9-mix and at 10 (i.e., 20%) and 50 (i.e., 37%) µg/L in the presence of S9-mix. Further, consideration of mean aberrant cell frequencies showed no real increases in culture tested with the test substance, when compared to control. Statistical analysis confirmed these observations. However, there was an apparent increase in the number of polyploid cells, compared to concurrent control values in all tested cultures, although a marked effect was seen only in activated cultures exposed to 50 µg/mL the author concluded that the test substance is not clastogenic in human lymphocytes under the experimental conditions. Under the study conditions, the test substance was not found to be clastrogenic in human lymphocytes (Richardson, 1989).

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From 21 August, 2006 to 01 November, 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted according to the OECD guideline 471 and Commission Directive 2000/32/EC, Annex-4D as well as in compliance with GLP. KL2 due to RA

Justification for type of information:

Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4D

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I (with and without S9 mix): 0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/plate
Experiment II (with and without S9 mix): 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: Deionised water was chosen because of its solubility properties and its low toxicity to the bacteria.

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

10 µg/plate for TA 1535 and TA 100 (Without metabolic activation)

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

Positive controls:

other:

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 10 µg/plate for TA 98, 50 µg/plate for TA 1537 (Without metabolic activation)

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

3 µL/plate for WP2 uvrA (Without metabolic activation)

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2.5 µg/plate for TA 1535, TA 1537, TA 98, TA 100 and 10 µg/plate for WP2 uvrA (With metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Triplicates

Evaluation criteria:

A test item is considered as a mutagen if there is a biologically relevant increase in the number of revertants exceeding the threshold by twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control.

A dose-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduction in the number of revertants observed in the test groups (i.e., at 100 – 333, 3 – 333 µg/plate with and without S9 mix respectively in experiment I; 100 – 333, 3 – 333 µg/plate respectively with and without S9 mix in experiment II)

Vehicle 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

Remarks:

Reduction in the number of revertants observed in the test groups (i.e., at 100 – 333, 3 – 333 µg/plate with and without S9 mix respectively in experiment I; 100 – 333, 3 – 333 µg/plate respectively with and without S9 mix in experiment II)

Vehicle 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

Remarks:

Reduction in the number of revertants observed in the test groups (i.e., at 100 – 333, 3 – 333 µg/plate with and without S9 mix respectively in experiment I; 100 – 333, 3 – 333 µg/plate respectively with and without S9 mix in experiment II)

Vehicle 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

Remarks:

Reduction in the number of revertants observed in the test groups (i.e., at 100 – 333, 3 – 333 µg/plate with and without S9 mix respectively in experiment I; 100 – 333, 3 – 333 µg/plate respectively with and without S9 mix in experiment II)

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduction in the number of revertants observed in the test groups (i.e., at 100 – 333, 3 – 333 µg/plate with and without S9 mix respectively in experiment I; 100 – 333, 3 – 333 µg/plate respectively with and without S9 mix in experiment II)

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the study conditions, the test substance was non-mutagenic in S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli strain WP2 uvrA in the reverse mutation assay with and without metabolic activation.

Executive summary:

An in vitro study was conducted to investigate the potential of read across substance, C16 TMAC (25% active in water) to induce gene mutations Salmonella typhimurium strains, according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA were treated with the read across substance using the Ames plate incorporation (pre-experiment and experiment I) and pre-incubation methods (experiment II). The assay was performed in three independent experiments both with and without metabolic activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The concentrations of the read across substance ranged from 3 to 5000 µg/plate in the pre-experiment, from 0.1 to 333 µg/plate in experiment I and from 0.01 to 333 µg/plate in experiment II. The plates incubated with the read across substance showed reduced background growth in all strains with and without metabolic activation in all experiments. Strong cytotoxic effects, evidenced by a reduction in the number of revertants, occurred in the test groups with and without S9 mix. No substantial increase in revertant colony numbers in any of the five tester strains was observed following treatment with the read across substance either in the presence or absence of S9 mix. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. All positive control showed a distinct increase in the number of revertant colonies. Under the study conditions, the substance was determined to be non-mutagenic with and without metabolic activation in the reverse mutation assay (Sokolowski, 2006). Based on the results of the read across study, similar result can be expected for the test substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Based on the results of the in vivo study, the test substance is considered to have no genotoxicity potential.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

micronucleus assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 15 February, 1983 to 18 March, 1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

not specified

Remarks:

pre-GLP

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River U.K. Limited, Margate, Kent
- Weight at study initiation: 18-21 g
- Assigned to test groups randomly: Yes
- Housing: Plastic disposable cage
- Diet: Spratt's Laboratory Diet number 1, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 10 d

ENVIRONMENTAL CONDITIONS
- Air changes (per hr): 30/h
- Photoperiod (hrs dark / hrs light): 12 h/12 h

Route of administration:

oral: gavage

Vehicle:

1% methyl cellulose

Details on exposure:

Dilution of the original solution were made in aqueous 1% methyl cellulose to give the desired concentration of the test substance. The mice were starved overnight prior to dosing. All animals in all groups were dosed with the standard volume of 2 mL/10 g bw. The test substance and vehicle control were dosed by oral gavage. The positive control was dosed by intraperitoneal injection.

Duration of treatment / exposure:

Single treatment

Frequency of treatment:

Single

Remarks:

Doses / Concentrations:
468 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

45 males and 45 females

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C was used. It was prepared as a solution in sterile 0.9% saline at a concentration of 0.4 mg/mL.

Tissues and cell types examined:

The femurs were cleared of tissue and one epiphysis removed from each bone.

Details of tissue and slide preparation:

A direct bone marrow smear was made on to a slide containing a drop of calf-serum. One smear was made from each femur. The prepared smears were air-dried and fixed in methanol. After fixation, the smears were air-dried and stained using Giemsa's technique. After rinsing in buffered distilled water, the slides were air-dried and mounted with coverslips using DPX. The stained smears were examined by light microscopy to determine the incidence of micronucleated cells/1,000 polychromatic erythrocytes/animal. The ratio of polychromatic to nonchromatic erythrocyte (NCEs) for each animal was assessed by examination of at least 1,000 erythrocyte.

Evaluation criteria:

Reproducible and significant increase in the number of micronucleated NCEs in the test group over that of the control group.

Statistics:

Non-parametric methods by Hollander M and Wolfe DA.

Key result

Sex:

male

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not clastogenic

Key result

Sex:

female

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not clastogenic

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
Probit analysis yielded an estimated LD10/3 of 468 mg/kg bw (LD50/3 was 884 mg/kg bw). A dose level of 468 mg/kg bw was chosen for the micronucleus test.

RESULTS OF DEFINITIVE STUDY- Induction of micronuclei (for Micronucleus assay): No significant difference as compared to controls.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of PCE to total erythrocytes remained essentially unaffected by the test substance
- Appropriateness of dose levels and route: Yes
- Statistical evaluation: Yes

No mortalities was seen in animals. Clinical signs included pilo-erection, hunched posture, lethargy and ptosis.

Conclusions:

Under the study conditions, the test substance was found to show no evidence of clastogenic potential in the bone marrow cells of mice.

Executive summary:

A study was conducted to determine the clastogenic potential of test substance, Coco TMAC (33% active in water) in an in vivo mouse bone marrow micronucleus test in mice, according to OECD 474 and EU Method B.12 Guidelines. Based on the results of a dose range finding assay, a dosage of 468 mg/kg bw (in1% methyl cellulose) was administered by oral gavage to male and female mice. Following dosing, the animals were examined regularly for any clinical signs of reaction. Bone morrow smears were obtained at 3 sampling times: 24, 48 or 72 hours after dosing. One smear from each animal was examined for the presence of micronuclei in 1000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A vehicle control (1% methylcellulose) and a psoitive control with mitomycin C by intraperitoneal injection were included. At all sampling times, mice treated with test substance showed no significant increase in the frequency of micronucleated polychromatic erythrocytes. There was no significant decrease in the ratio of polychromatic to normochromatic erythrocytes at any of the three kill times after treatment. The positive control compound, mitomycin C, produced large, highly significant increases in the frequency of micronucleated polychromatic erythrocytes together with large decreases in the ratio of polychromatic to normochromatic erythrocytes and increases in the frequency of micronucleated normochromatic erythrocytes. Under the study conditions, the test substance was found to show no evidence of clastogenic potential in the bone marrow cells of mice (Allen, 1983).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro 

Study 1:An in vitro study was conducted to determine the mutagenic potential of test substance, TMAC C (33% active in water), according to OECD 471 and EPA OPPTS 870.5265 Guidelines, in compliance with GLP. Using pour-plate assays, the test substance was examined for mutagenic activity in four strains of Salmonella typhimurium: TA 100, TA 1535, TA 1537 and TA 98 in the absence and in the presence of S9-mix. The test concentrations included 0, 1, 3, 10, 32 and 100 µg/plate (i.e., equivalent to 0, 0.33, 0.99, 3.3, 10.56 and 33 µg/plate). No increase in reversion to prototrophy was obtained with any of the bacterial strain at any concentration level either in the presence or absence of S9-mix. Further, inhibition of growth, observed as thinning of the background lawn of non-revertant cells, occurred in all strains following exposure to the test substance at 100 µg/plate.Appropriate positive control chemicals (with S-9 mix where required) induced marked increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S-9 mix. Under the study conditions, the test substance was not mutagenic with and without metabolic activation (May, 1989). 

Study 2:Anin vitrostudy was conducted to investigate the potential of read across substance, C16 TMAC (25% active in water) to induce gene mutations Salmonella typhimurium strains, according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA were treated with the read across substance using the Ames plate incorporation (pre-experiment and experiment I) and pre-incubation methods (experiment II). The assay was performed in three independent experiments both with and without metabolic activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The concentrations of the read across substance ranged from 3 to 5000 µg/plate in the pre-experiment, from 0.1 to 333 µg/plate in experiment I and from 0.01 to 333 µg/plate in experiment II. The plates incubated with the read across substance showed reduced background growth in all strains with and without metabolic activation in all experiments. Strong cytotoxic effects, evidenced by a reduction in the number of revertants, occurred in the test groups with and without S9 mix. No substantial increase in revertant colony numbers in any of the five tester strains was observed following treatment with the read across substance either in the presence or absence of S9 mix. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. All positive control showed a distinct increase in the number of revertant colonies. Under the study conditions, the substance was determined to be non-mutagenic with and without metabolic activation in the reverse mutation assay (Sokolowski, 2006). Based on the results of the read across study, similar result can be expected for the test substance.

Study 3:Anin vitro study was conducted to determine the clastogenic potential of the test substance, TMAC C (33% active), in cultured human lymphocytes according to OECD Guideline 473, in compliance with GLP. After a concentration range finding test, two independent tests were performed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (S9-mix). With S9-mix cells were exposed for 24 hours. The test substance produced a reduction in mitotic activity (i.e., approximately 48%) at 10 µg/L in the absence of S9-mix and at 10 (i.e., 20%) and 50 (i.e., 37%) µg/L in the presence of S9-mix. Further, consideration of mean aberrant cell frequencies showed no real increases in culture tested with the test substance, when compared to control. Statistical analysis confirmed these observations. However, there was an apparent increase in the number of polyploid cells, compared to concurrent control values in all tested cultures, although a marked effect was seen only in activated cultures exposed to 50 µg/mL the author concluded that the test substance is not clastogenic in human lymphocytes under the experimental conditions. Under the study conditions, the test substance was not found to be clastogenic in human lymphocytes (Richardson, 1989).   

Study 4:Anin vitrostudy was conducted to determine the potential of the test substance, TMAC C (35.5% active in water), to induce mutations at the TK (thymidine kinase) locus in L5178Y mouse lymphoma cells. Study was performed according to OECD 476 Guideline, EU Method B.17 and Commission directive 2002/32/EC and U.K Environmental Mutagen Society, in compliance with GLP. After a preliminary toxicity test, 0.625, 1.25, 2.5, 5, 10 and 20 µg/L was selected as the test concentrations for the first experiment. The experiment was repeated to confirm the results of the first experiment. Three-hour exposures were used both with and without S9-mix while for the second experiment, the exposure time was increased to 24 h. For the second experiment, the concentration range was 2.5 to 30 µg/L with S9-mix and 0.313 to 5 µg/L without S9-mix. The test substance did not induce a statistically significant or concentration-related increase in the mutant frequency at any concentration level, either with or without metabolic activation. Adequate levels of toxicity were achieved in all exposure groups. Under the study conditions, the test substance was not found to show mutagenic activity in mouse lymphoma assay (Nolan, 2002).   

In vivo 

A study was conducted to determine the clastogenic potential of test substance, TMAC C (33% active in water) in anin vivomouse bone marrow micronucleus test in mice, according to OECD 474 and EU Method B.12 Guidelines. Based on the results of a dose range finding assay, a dosage of 468 mg/kg bw (in1% methyl cellulose) was administered by oral gavage to male and female mice. Following dosing, the animals were examined regularly for any clinical signs of reaction. Bone morrow smears were obtained at 3 sampling times: 24, 48 or 72 hours after dosing. One smear from each animal was examined for the presence of micronuclei in 1000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A vehicle control (1% methylcellulose) and a psoitive control with mitomycin C by intraperitoneal injection were included. At all sampling times, mice treated with test substance showed no significant increase in the frequency of micronucleated polychromatic erythrocytes. There was no significant decrease in the ratio of polychromatic to normochromatic erythrocytes at any of the three kill times after treatment. The positive control compound, mitomycin C, produced large, highly significant increases in the frequency of micronucleated polychromatic erythrocytes together with large decreases in the ratio of polychromatic to normochromatic erythrocytes and increases in the frequency of micronucleated normochromatic erythrocytes. Under the study conditions, the test substance was found to show no evidence of clastogenic potential in the bone marrow cells of mice (Allen, 1983).  

The biocide assessment report available from RMS Italy on TMAC C, assessed the endpoint based on the above studies submitted by Akzo Nobel (now Nouryon) and additional studies submitted on TMAC C by Lonza Cologne GmbH. The below summary was presented for the studies submitted by Lonza: 

“ATMAC displayed no genotoxic activity in the three mutagenicity tests required for the authorisation of Biocidal Products: Salmonella mutagenicity assay (in TA1535, TA1537, TA98 and TA100 strains), gene mutation study in mouse lymphoma L5178Y cells and in-vitro cytogenetic test in human lymphocytes. Moreover, no induction of micronuclei was observed in a mouse bone marrow micronucleus test, following oral administration of the test substance (Lonza Cologne GmbH). In the in-vitro cytogenetic test in human lymphocytes polyploidy was incidentally observed, with a dose-dependent trend only in the presence of metabolic activation. This phenomenon might reflect spindle function disturbance, possibly resulting in the induction of numerical aberrations. If this is the case, a positive result in the in vivo micronucleus test would be expected. Micronucleus assay, correctly performed at the MTD, resulted negative. At this dosage animals showed evident clinical signs but no local toxicity (alteration in the ratio (PCE/NCE), therefore there was no direct evidence that the drug has actually reached the target organ (Lonza Cologne GmbH). In conclusion, the test substance resulted negative in all the required genotoxicity studies and appeared unable to directly damage DNA.”   

Overall, it was concluded:“According to the studies presented by both applicants, genotoxic potential in vitro and in vivo of coco alkyltrimethylammonium chloride can be ruled out”(ECHA biocides assessment report, 2016).  

Therefore, based on the results from the availablein vitroandvivogenotoxicity studies, as well as in line with the biocides assessment report, the test substance is concluded to have no genotoxicity potential.  

Justification for classification or non-classification

Based on the available negative results from in vitro and in vivo studies, the test substance does not warrant a classification for genotoxicity according to the EU CLP (Regulation 1272/2008/EC) criteria.