Quaternary ammonium compounds, C18-22 (even numbered) alkyltrimethyl, chlorides; Docosyltrimethylammonium chloride; C18-22 TMAC

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of the read across studies, the test substance C18-22 TMAC is considered as non-genotoxic.

Link to relevant study records

Reference

Reference 1

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:

1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: See remarks

Remarks:

Study was performed according to OECD guideline published at that time and according to GLP. E. coli WP2 strain was not used. The second experiment was performed as a plate incorporation assay, but not as a preincubation assay. Since the effects were clearly negative, these limitations are not considered to decisively limit the reliability of the study.

Justification for type of information:

Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification. The 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 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Pre-Experiment and Experiment I: 0 (control), 4, 20, 100, 500, 2500, 5000 µg/plate
Experiment II: 0 (control), 4, 20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene (with metabolic activation for all strains)

Remarks:

See below for additional information

Details on test system and experimental conditions:

The assay was performed in two independent experiments:

experiment I: plate incorporation assay with and without induced rat liver S9 mix
experiment II: plate incorporation assay with and without induced rat liver S9 mix

Rat liver S9 mix from Aroclor 1254 induced rats was used.

Top agar was prepared for the Salmonella strains by mixing 100 ml agar (0.6% (w/v) agar, 0.5% (w/v) NaCI) with 10 ml of a 0.5 mM histidine-biotin solution. After mixing, the liquid was poured into a petridish with minimal agar (1.5 % (w/v) agar, Vogel-Bonner E medium with 2 % (wlv) glucose). After incubation for approximately 48 hours at approx. 37 °C in the dark, colonies (hi? revertants) were counted.

DURATION
- Exposure duration: after solidification the plates were incubated upside down for approx. 48 hours at 37°C in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: The first experiment was performed with all tester strains using three plates per dose to get information on mutagenicity and toxicity for calculating an appropriate dose range. A reduced rate of spontaneously occuring colonies and visible thinning of the bacterial lawn were used as toxicity indicators. Thinning of the bacterial lawn was evaluated microscopically.
In combination with the second experiment, toxicity testing was performed as follows: 0.1 ml of the different dilutions of the test compound were thoroughly mixed with 0.1 ml of 108 dilution of the overnight culture of TA 100 and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control is compared with the number of colonies per plate in the presence of the test compound. Results are given as a ratio of these values (= surviving fraction).

POSITIVE CONTROL SUBSTANCES:
without metabolic activation: sodium azide (TA 1535, TA 100), 9-aminoacridine (TA 1537), 2-nitrofluorene (TA 98);
with metabolic activation: 2-aminoanthracene (all strains)

Evaluation criteria:

A test substance is classified as mutagenic if it has either of the following effects:

a) a test substance produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn

b) a test substance induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test substance at complete bacterial background lawn.

The test results must be reproducible.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See below for additional information

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test substance was tested at doses of 4 to 5000 microgram/plate and proved to be toxic to most of the bacterial strains at doses of 2500 microgram/plate and above. Thinning of the bacterial lawn and a reduction in the number of colonies were observed at this dose.

Remarks on result:

other: all strains/cell types tested

Mean mutant number

Exp. I: plate incorporation method without S9 mix

Concentrations given in µg/plate

Strain -- 0 -- 4 -- 20 -- 100 -- 500 -- 2500 -- 5000

TA100 -- 176.7 -- 168.0 -- 201.0 -- 217.0 -- 25.0 -- 1.3 -- 1.0

TA1535 -- 10.7 -- 6.3 -- 8.3 -- 8.0 -- 1.3 -- 1.0 -- 0.3

TA1537 -- 7.0 -- 10.7 -- 9.7 -- 11.3 -- 5.0 -- 0.0 -- 0.0

TA98 -- 27.7 -- 24.0 -- 31.3 -- 23.0 -- 7.3 -- 1.0 -- 0.0

Exp. I: plate incorporation method with rat S9 mix

Concentrations given in µg/plate

Strain -- 0 -- 4 -- 20 -- 100 -- 500 -- 2500 -- 5000

TA100 -- 137.7 -- 207.0 -- 182.7 -- 185.0 -- 165.7 -- 43.0 -- 6.0

TA1535 -- 9.3 -- 11.7 -- 14.7 -- 12.3 -- 10.0 -- 5.0 -- 1.0

TA1537 -- 11.7 -- 8.7 -- 10.0 -- 8.3 -- 6.3 -- 2.3 -- 0.7

TA98 -- 36.3 -- 34.7 -- 36.7 -- 41.7 -- 37.0 -- 15.0 -- 2.0

Exp. II: plate incorporation method without S9 mix

Concentrations given in µg/plate

Strain -- 0 -- 4 -- 20 -- 100 -- 500 -- 2500 -- 5000

TA100 -- 141.7 -- 184.3 -- 146.3 -- 145.7 -- 2.0 -- 1.0 -- 1.0

TA1535 -- 16.0 -- 12.7 -- 7.7 -- 8.7 -- 3.3 -- 0.0 -- 0.0

TA1537 -- 7.7 -- 7.7 -- 8.7 -- 8.3 -- 4.3 -- 0.0 -- 0.0

TA98 -- 26.3 -- 24.3 -- 22.0 -- 31.0 -- 9.3 -- 1.3 -- 0.0

Exp. II:

plate incorporation method with rat liver S9 mix

Concentrations given in µg/plate

Strain -- 0 -- 4 -- 20 -- 100 -- 500 -- 2500 -- 5000

TA100 -- 176.3 -- 172.7 -- 170.3 -- 170.3 -- 123.3 -- 98.7 -- 24.0

TA1535 -- 19.3 -- 13.0 -- 16.7 -- 13.3 -- 15.0 -- 1.3 -- 1.0

TA1537 -- 8.0 -- 7.0 -- 9.7 -- 10.3 -- 6.0 -- 9.3 -- 0.3

TA98 -- 36.7 -- 33.3 -- 36.3 -- 29.3 -- 30.7 -- 8.3 -- 1.3

Conclusions:

Based on the results of the read across study, similar absence of mutagenic potential is expected for the test substance, C18-22 TMAC, in an Ames test.

Executive summary:

An in vitro study was conducted to determine the mutagenic potential of the read across substance, C20-22 TMAC (active content not specified), using bacterial reverser mutation assay (Ames test), according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA 98, TA100, TA 1535 and TA 1537 was used in this experiment. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, i.e., the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the read across substance. The plates were inverted and incubated for 48 h at 37±2°C in dark, colonies were counted. The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. The read across substance was dissolved in ethanol and 6 test concentrations ranging from 4 μg /plate to 5000 μg/plate (i.e., 0, 4, 20, 100, 500, 2500 and 5000 μg per plate) was used. The number of spontaneous revertant colonies in the control plates (without the mutagen) was similar to that described in the literature.Toxicity was observed at 2500 μg/plate and above in the bacterial strains; therefore 5000 μg/plate was chosen as top dose level for the mutagenicity study. The read across substance did not show a dose dependent increase in the number of revertants in any of the bacterial strains in the presence or absence of metabolic activation system. The negative and positive controls gave results within the expected range; hence the experiment was considered valid. Under the study conditions, the read across substance was determined to be non-mutagenic in Ames test, with or without metabolic activation (Muller, 1995). Based on the results of the read across study, similar absence of mutagenic potential is expected for the test substance, C18-22 TMAC, in an Ames test.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Study period:

From July, 2006 to March, 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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. The 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 (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation test using the Hprt and xprt genes (migrated information)

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: minimum essential medium containing 10% fetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver protein with cofactors)

Test concentrations with justification for top dose:

Doses applied in the gene mutation assay with Behentrimonium chloride (related to active substance) as concentration in μg/mL

Experiment I
without S9 mix 0.1, 0.2, 0.3, 0.6, 1.3, 1.9
with S9 mix 1.3, 2.4, 5.0, 9.9, 19.7, 29.6

Experiment II
without S9 mix 0.6, 1.2, 2.4, 4.7, 6.3, 7.9

Vehicle / solvent:

Water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (4h-incubation without serum, 24 h incubations with serum)


DURATION
- Preincubation period: none
- Exposure duration: 4 and 24 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days


SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution


NUMBER OF REPLICATIONS: 5/experiment, 2 experiments plus repetition of the 1st experiment

Positive control substances: ethylmethane sulfonate without metabolic activation and 7,12-Dimethylbenz(a)anthracene with metabolic activation
Metabolic activation: liver S9 was prepared from phenobarbital-treated rats


NUMBER OF CELLS EVALUATED: colonies >50 cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test substance is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.

The test substance is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.5 – 31.8 mutants per 106 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of negative and solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT® statistics software. The number of mutant colonies obtained in the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance should be considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See text below

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: negligible increase at highest dose (pH 7.45 at 78.8 µg/ml vs. 7.37 in solvent control
- Effects of osmolality: negligible: 284 mOsm at highest dose of 78.7 µg/ml vs. 285 in solvent control

Cytotoxicity:

Relevant toxic effects indicated by a relative cloning efficiency I (CE I) below 50% occurred in the first experiment at 1.3 μg/mL and above without metabolic activation and at 9.9 (culture I only) and 19.7 μg/mL and above in the presence of metabolic activation. In the second experiment such toxic effects occurred at 4.7 μg/mL and above. The cultures at 29.6 μg/mL in the first experiment with metabolic activation and at 6.3 and 7.9 μg/mL in the second experiment were not analysable due to exceedingly severe toxic effects. The CE I was zero or well below 10 % at 9.9 (culture I only) and 19.7 μg/mL in the first experiment with and at 1.9 μg/mL without metabolic activation. Still, the data are considered valid since the corresponding cell density of the mass cell cultures used to determine mutagenicity determined at the first sub-cultivation following treatment was above 20 % of the corresponding control value. In the second experiment severe toxicity resulted in CE values of 0 at 4.7 μg/mL and corresponding cell densities of 6.5 % and 9.8 %. Still, the surviving cells recovered and showed cloning efficiency II values of more than 50 % at the time point of selection. The fact, that higher concentrations are tolerated in the second experiment compared to the first experiment without metabolic activation indicates possible protein or lipid binding effects of the test substance. The FCS-concentration is 10 % during long term treatment compared to 0 % during 4 h treatment. Test substances binding to proteins or lipids of the FCS are less available to the cells and thus, higher concentrations are tolerated even though the treatment period is much longer.

 

No relevant and reproducible increase of the mutation frequency occurred at any concentration with and without metabolic activation. All mutant frequencies remained well within the historical range of negative and solvent controls. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was calculated in both main experiments.

In both experiments of this study (with and without S9 mix) the range of the negative and solvent controls was from 2.9 up to 10.1 mutant colonies per 106 cells; the range of the groups treated with the test substance was from 1.2 up to 12.4 mutant colonies per 106 cells. EMS (0.300 mg/mL in experiment I and 0.150 mg/mL in experiment II) and DMBA (1.5 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

 

Summary of results

Condition -- Conc. (µg/mL) -- Rel. cloning efficiency I (%) -- Cell density (% of control) -- Mutant colonies per 10e6 cells -- Induction factor

 

Exp. I, 4 h treatment without S9 mix, Culture 1

Negative control -- -- 100.0 -- 100.0 -- 6.9 --

Solvent control with water -- -- 100.0 -- 100.0 -- 6.4 --1.0

Positive control with EMS -- 300.0 -- 87.6 -- 116.0 -- 119.5 -- 17.2

Test substance -- 0.1 -- 92.8 -- culture was not continued

Test substance -- 0.2 -- 100.8 -- 103.0 -- 2.0 -- 0.3

Test substance -- 0.3 -- 99.5 -- 92.4 -- 5.6 -- 0.9

Test substance -- 0.6 -- 98.3 -- 80.6 -- 12.4 -- 2.0

Test substance --1.3 -- 22.7 -- 80.6 -- 2.1 -- 0.3

Test substance -- 1.9 -- 0.0 -- 34.9 -- 3.4 -- 0.5

 

Exp. I, 4 h treatment without S9 mix, Culture 2

Negative control -- -- 100.0 -- 100.0 -- 4.8 --

Solvent control with water -- -- 100.0 -- 100.0 -- 2.9 --1.0

Positive control with EMS -- 300.0 -- 85.3 -- 96.6 -- 94.0 -- 19.6

Test substance -- 0.1 -- 85.9 -- culture was not continued

Test substance -- 0.2 -- 87.9 -- 111.0 -- 1.5 -- 0.5

Test substance -- 0.3 -- 85.8 -- 122.9 -- 3.7 -- 1.3

Test substance -- 0.6 -- 76.8 -- 113.4 -- 7.3 -- 2.5

Test substance --1.3 -- 21.5 -- 78.1 -- 4.8 -- 1.6

Test substance -- 1.9 -- 0.0 -- 30.2 -- 1.5 -- 0.5

 

 

Exp. I, 4 h treatment with S9 mix, Culture 1

Negative control -- -- 100.0 -- 100.0 -- 4.2 --

Solvent control with water -- -- 100.0 -- 100.0 -- 3.6 --1.0

Positive control with DMBA -- 1.5 -- 62.1 -- 67.4 -- 931.2 -- 219.6

Test substance -- 1.3 -- 87.2 -- 77.3 -- 1.2 -- 0.3

Test substance -- 2.4 -- 82.5 -- 78.4 -- 1.4 -- 0.4

Test substance -- 5.0 -- 75.5 -- 58.6 -- 7.9 -- 2.2

Test substance -- 9.9 -- 3.6 -- 34.5 -- 4.4 -- 1.2

Test substance --19.7 -- 0.0 -- 20.0 -- 3.6 -- 1.0

Test substance -- 29.6 -- 0.0 -- culture was not continued

 

Exp. I, 4 h treatment with S9 mix, Culture 2

Negative control -- -- 100.0 -- 100.0 -- 7.7 --

Solvent control with water -- -- 100.0 -- 100.0 -- 10.1 --1.0

Positive control with DMBA -- 1.5 -- 21.2 -- 46.4 -- 917.6 -- 119.7

Test substance -- 1.3 -- 97.9 -- 96.8 -- 7.8 -- 0.8

Test substance -- 2.4 -- 104.6 -- 102.9 -- 7.8 -- 0.8

Test substance -- 5.0 -- 93.4 -- 61.6 -- 1.4 -- 0.1

Test substance -- 9.9 -- 78.5 -- 58.0 -- 4.6 -- 0.5

Test substance -- 19.7 -- 5.1 -- 26.5 -- 3.7 -- 0.4

Test substance -- 29.6 -- 0.6 -- culture was not continued

 

 

Exp.II, 24 h treatment without S9 mix, Culture 1

Negative control -- -- 100.0 -- 100.0 -- 3.7 --

Solvent control with water -- -- 100.0 -- 100.0 -- 3.3 --1.0

Positive control with EMS -- 150.0 -- 72.2 -- 101.8 -- 184.8 -- 49.6

Test substance -- 0.6 -- 98.1 -- 96.6 -- 7.4 -- 2.3

Test substance -- 1.2 -- 91.5 -- 78.0 -- 4.6 -- 1.4

Test substance -- 2.4 -- 64.7 -- 30.3 -- 6.9 -- 2.1

Test substance -- 4.7 -- 0.0 -- 6.5 -- 2.7 -- 0.8

Test substance -- 19.7 -- 0.0 -- culture was not continued

Test substance -- 29.6 -- 0.0 -- culture was not continued

 

Exp.II, 24 h treatment without S9 mix, Culture 2

Negative control -- -- 100.0 -- 100.0 -- 3.5 --

Solvent control with water -- -- 100.0 -- 100.0 -- 5.4 --1.0

Positive control with EMS -- 150.0 -- 72.9 -- 117.6 -- 210.1 -- 60.5

Test substance -- 0.6 -- 104.3 -- 75.3 -- 6.8 -- 1.3

Test substance -- 1.2 -- 94.4 -- 73.5 -- 4.2 -- 0.8

Test substance -- 2.4 -- 74.3 -- 37.5 -- 8.3 -- 1.6

Test substance -- 4.7 -- 0.0 -- 9.8 -- 7.8 -- 1.5

Test substance -- 19.7 -- 0.0 -- culture was not continued

Test substance -- 29.6 -- 0.0 -- culture was not continued

 

where indicated low concentration cultures were not continued since a minimum of only four analysable concentrations is required, while high concentration cultures were not continued due to exceedingly strong toxic effects

Conclusions:

Under the study conditions, the test substance was not found to induce gene mutations in the HPRT locus in V79 Chinese hamster cells, either in the presence or absence of metabolic activation.

Executive summary:

An in vitro study was conducted to investigate the potential of the read across substance, C22 TMAC (active: 78.8%), to induce gene mutations in V79 Chinese hamster cells, according to OECD Guideline 476, in compliance with GLP. The target for gene mutation was the gene coding for hypoxanthine-guanine phosphoribosyl transferase (HPRT), an enzyme that can convert 6-thioguanine (6-TG) to its toxic ribophosphorylated derivative. Chinese Hamster V79 cells were exposed to the read across substance over a period of either 4 or 24 h (for the 4 hours exposure, with and without metabolic activation by rat liver S9 mix). After exposure, cells were maintained for 7 d read across substance-free under normal growth conditions to deplete HPRT in mutated cells. After this period, cells were maintained for 8 d in 6-TG containing medium, i.e., toxic to non-mutated (HPRT-expressing) cells. After this selection period, the number of colonies remaining (each representing a mutated cell) was counted and their number was compared to those achieved in the solvent controls and positive control substances, known to be mutagenic. Relevant toxic effects indicated by a relative cloning efficiency I (CE I) below 50% occurred in the first experiment at 1.3 μg/mL and above without metabolic activation and at 9.9 (culture I only) and 19.7 μg/mL and above in the presence of metabolic activation. In the second experiment such toxic effects occurred at 4.7 μg/mL test concentration and above. No relevant and reproducible increase of the mutation frequency occurred at any test concentration with and without metabolic activation. All positive controls showed a distinct increase in the number of mutant colonies. All mutant frequencies remained well within the historical range of negative and solvent controls; hence the experiment was considered valid. Under the study conditions, the read across substance was not found to induce gene mutations in the HPRT locus in V79 Chinese hamster cells, either in the presence or absence of metabolic activation (Wollny, 2007).Based on the results of the read across study, a similar absence of mutagenic potential is expected for the test susbtance, C18-22 TMAC in the HPRT assay.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: According to Draft Proposal for a new Guideline No. 487

Remarks:

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. The 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 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks (Greiner, 72632 Frickenhausen, Germany). About 5 x 105 cells per flask were seeded in 15 mL of MEM (minimal essential medium; Seromed, 12247 Berlin, Germany) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, 35091 Cölbe, Germany). The cells were subcultured twice weekly. The cell cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5% air).

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

Exp. I: with and without S9 mix: 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, 50.0, 100.0, 200.0, and 400.0 µg/mL
Exp. IIA: with and without S9 mix: 0.2, 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, and 25.0 µg/mL
Exp. IIB: without S9 mix: 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10.0, 12.5, and 15.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Water

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

Two independent experiments were performed.
In Experiment I the exposure period was 4 h with and without metabolic activation.
In Experiment II the exposure period was 20 h without S9 mix and 4 h with metabolic activation.

The cells were prepared 24 h (48 h for Exp. II with S9 mix) after start of treatment with the test substance.

METHOD OF APPLICATION: in minimal essential medium

DURATION
- Exposure duration: 4 and 24 h
- Expression time (cells in growth medium): 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h (48 h for Exp. II with S9 mix)

SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays): May Gruenwald and Giemsa

NUMBER OF REPLICATIONS: 1.5 - 2

NUMBER OF CELLS EVALUATED: 2000

EVALUATION: Evaluation of the cultures was performed manually using NIKON microscopes with 40 x oil immersion objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei were stained in the same way as the main nucleus. The area of the micronucleus did not extend the third part of the area of the main nucleus. 2000 cells were scored per test group. The frequency of micronucleated cells was reported as % micronucleated cells.

DETERMINATION OF CYTOTOXICITY
- Method: Proliferation Index

OTHER EXAMINATIONS:

Evaluation criteria:

A test substance can be classified as mutagenic if:

- the number of micronucleated cells is not in the range of the historical control data (0.0-2.0% micronucleated cells), and either a concentration-related increase in three test groups or a significant increase of micronucleated cells in at least one test group is observed.

A test substance can be classified as non-mutagenic if:
- The number of micronucleated cells in all evaluated test groups is in the range of the historical control data (0.0-2.0% micronucleated cells), and/o no concentration-related increase in the number of micronucleated cells is observed.

Statistical significance can be confirmed by means of the Chi square test. However, both biological and statistical significance should be considered together. If the criteria above mentioned for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Statistics:

Statistical significance can be confirmed by means of the Chi square test.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See text below

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test substance, suspended (Experiment I) or dissolved (Experiments IIA and llB) in deionised water, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vifro in the absence and the presence of metabolic activation by S9 mix.
Three independent experiments were performed. In Experiment I, the exposure period was 4 hs with and without metabolic activation. In Experiment IIA and IIB, the exposure period was 20 hrs without S9 mix and in Experiment 11, the exposure period was 4 hs with metabolic activation. The cells were prepared 24 hs (Exp. I, IIA and llB) and 48 hrs (Exp. IIA) after start of treatment with the test substance.
In each experimental group two parallel cultures were set up. Per culture 1000 cells were scored for micronuclei.
Evaluated experimental points after treatment with test substance:
Exp I: 3.1, 6.3 and 12.5 µg/ml, without S9 mix, 4 hrs exposure, 24 hrs preparation interval
Exp IIA: 3.1, 6.3 and 12.5 µg/ml, without S9 mix, 20 hrs exposure, 24 hrs preparation interval
Exp IIB: 5.0, 6.0, and 7.0 µg/ml, without S9 mix, 20 hrs exposure, 24 hrs preparation interval
Exp I: 6.3, 12.5 and 25.0 µg/ml, with S9 mix, 4 hrs exposure, 24 hrs preparation interval
Exp IIA: 3.1, 6.3 and 12.5 µg/ml, with S9 mix, 4 hrs exposure, 48 hrs preparation interval

In Experiment I, no relevant increase of the pH value or osmolarity was observed (solvent control 271 mOsm, pH 7.3 versus 288 mOsm and pH 7.4 at 400.0 µg/mL). Test substance precipitation was observed at a concentration of 50.0 µg/mL and above in the absence of S9 mix and at 25.0 µg/mL and above in the presence of S9 mix. Clear toxic effects indicated by reduced cell numbers were observed after treatment with 12.5 µg/mL (11.3 % of control) and above in the absence of S9 mix. In the presence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage.
In the absence and presence of S9 mix, no biologically relevant increase in the percentage of micronucleated cells was observed up to the highest scorable concentration. The rates of micronucleated cells after treatment with the test substance in the absence of S9 mix (0.95 - 1.55 %) and in the presence of S9 mix (0.85 - 1.55 %) were below the corresponding solvent control values (2.00 and 1.70 %, respectively) and within the range the laboratory's historical control data: 0.0 - 2.0 % micronucleated cells.
In Experiment IIA, test substance precipitation was observed at a concentration of 6.3 µg/mL and above in the absence of S9 mix and at 25.0 µg/mL and above in the presence of S9 mix. Clear cytototoxicity (indicated by reduced cell numbers when compared to control values) of about 60% or above were observed after treatment with 12.5 µg/mL (70 % cytotoxicity) and above in the absence of S9 mix, and with 12.5 µg/mL (58 % cytotoxicity) and above in the presence of S9 mix.
In the presence of S9 mix, no biologically relevant increase in the percentage of micronucleated cells was observed up to the highest evaluated concentration of 12.5 µg/mL. The rates of micronucleated cells after treatment with the test substance (0.60 - 1.00%) were close to the corresponding solvent control value (0.55 %) and within the range of our historical control data: 0.0 - 2.0 % micronucleated cells. In the absence of S9 mix, dose-related increases in the percentage of micronucleated cells (1 -65 %, 1.75 %, and 2.40 %) were observed at the three evaluated concentrations (3.1, 6.3, and 12.5 µg/mL, respectively). Although all three values were statistically significantly increased compared to the corresponding solvent control value (0.85%), the percentages of micronucleated cells at 3.1 and 6.3 µg/mL (1.65 % and 1.75 %, respectively) were within the laboratorie's historical control data range (0.0 - 2.0 % micronucleated cells), and these increases have to be regarded as biologically irrelevant. At the highest concentration evaluated (12.5µg/mL), given the high cytotoxicity observed (70%), the biological relevance of the increased incidence of micronucleated cells remained ambiguous.
Accordingly, two repeat experiments (Experiment IIB) within a narrow concentration range were performed, in order to verify the observation in Experiment IIA.
The first repeat experiment could not be evaluated, since only two concentrations were available for cytogenetic evaluation, which is not in compliance with the guideline recommendations. In both experiments, concentrations of 7.5 µg/mL and 8.0 µg/mL and above were not scorable for cytogenetic damage due to strong test substance induced cytotoxicity.
In the second repeat experiment, test substance precipitation was observed at a concentration of 6.0 µglmL and above in the absence of S9 mix. Clear toxic effects indicated by reduced cell numbers were observed after treatment with 7.0 µg/mL (62 % cytotoxicity). No biologically relevant increase in the percentage of micronucleated cells was observed up to the highest scorable concentration. The rates of micronucleated cells after treatment with the test substance (0.05 - 1.05 %) were close to the corresponding solvent control values (0.55 %) and within the range of our historical control data: 0.0 - 2.0 % micronucleated cells. Accordingly, the increased incidence of micronucleated cells observed at a concentration associated with high cytotoxicity
(Experiment IIA in the absence of S9 mix) could not be reproduced, and this finding was considered to bear no biological significance.
In Experiment IIB, test substance precipitation was observed at a concentration of 6.0 µg/mL and above in the absence of S9 mix. Clear toxic effects indicated by reduced cell numbers were observed after treatment with 7.0 µg/mL.
Colcemid (25 ng/mL to 10 µg/mL), Mitomycin C (0.03 or 0.1 µg/mL) or CPA (2.5 to 25 µg/mL) were evaluated as positive controls and showed a distinct increase in the percentage of micronucleated cells.
In conclusion, it can be stated that under the experimental conditions reported, the test substance Test substancedid not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and the presence of metabolic activation.
Therefore, Test substancehas to be considered as non-mutagenic in this in vitro test system, when tested up to cytotoxic test substance concentrations (Experiment I, with metabolic activation, Experiment IIA with and without metabolic activation, and Experiment IlB, without metabolic activation) or the highest scorable concentration (Experiment I, with metabolic activation).

Remarks on result:

other: all strains/cell types tested

Dose selection:

Dose selection was performed following the current OECD Guideline for in vitro chromosome aberration studies (OECD Guideline no. 473). The highest concentration chosen for the evaluation of genotoxicity should produce clear toxicity with reduced cell growth, determined by the mean of the cell count prior to cell seeding on slides, by > 60 % and/or the occurrence of precipitation. In case of nontoxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, if formulability in an appropriate solvent is possible.

With respect to the molecular weight of the test test substance, 5000.0 µg/mL of Test substance (approx. 9.7 mM) were applied as top concentration in Experiment I. Test test substance concentrations between 9.8 and 5000.0 µg/mL (with and without S9 mix) were chosen for the treatment of the cultures. Due to strong toxic effects indicated by reduced cell numbers at test test substance concentrations of 9.8 µg/mL and above in the absence and presence of metabolic activation, Experiment I was repeated with test test substance concentrations between 0.8 and 400.0 µg/mL (with and without S9 mix). With regard to the purity of the test test substance, the corresponding amount of the active substance in the respective test test substance concentrations was 78.8 %.

In Experiment I, precipitation of the test test substance in culture medium was observed with 50.0 µg/mL and above in the absence of S9 mix, and with 25.0 µg/mL and above in the presence of S9 mix. Using reduced cell numbers of about 40 % of control or below as an indicator for toxicity in Experiment I, clear toxic effects were observed after 4 hrs treatment with 12.5 µg/mL and above in the absence of S9 mix, and with 50.0 µg/mL and above in the presence of S9 mix. With respect to the results obtained in Experiment I, 25.0 µg/mL was chosen as top concentration for Experiment IIA in the absence and presence of S9 mix. In a confirmatory experiment, designated Experiment llB, concentrations between 2.5 and 20.0 µg/mL, in the absence of S9 mix, were chosen in order to verify the results obtained in Experiment IIA. Due to strong test test substance induced cytotoxicity at concentrations of 7.5 µg/mL and above, only two concentrations were scorable for cytogenetic damage. Therefore, this experimental part was repeated with test test substance concentrations between 0.5 and 15.0 µg/mL.

Summary of results of the micronucleus test with test substance.

Summary of results of the micronucleus test with Behentrimonium chloride

Exp.	Preparation	Test system	Cell number	Micronucleated
	interval	concentration	In %	cells
		in µg/mL	of control	in %
Exposure period 4 hrs without S9 mix
I	24 h	Negative control	103	0.55
		Solvent control^1	100	2.00
		Positive control^2	78.9	5.1S
		Positive control^3	80.2	3.25S
		Positive control^4	78.1	4.70S
		Positive control^5	45.8	17.95S
		3.1	88.9	0.95
		6.3	62.7	1.55
		12.5	11.3	1.15
Exposure period 20 hrs without S9 mix
IIA	24 h	Negative control	99.5	1.05
		Solvent control^1	100	0.85
		Positive control^2	51.8	28.3S
		Positive control^3	44.4	28.80S
		Positive control^4	72.3	9.45S
		Positive control^5	61.4	13.85S
		3.1	97.4	1.65S
		6.3 P	57.5	1.75S
		12.5 P	29.9	2.40S
Exposure period 20 hrs without S9 mix
IIB	24 h	Negative control	90.0	0.30
		Solvent control^1	100	0.55
		Positive control^2	40.6	66.35S
		Positive control^3	33.8	50.35S
		Positive control^4	64.0	6.85S
		Positive control^5	69.8	6.00S
		5.0	66.4	1.05
		6.0 P	47.1	0.70
		7.0 P	37.5	0.05
Exposure period 4 hrs with S9 mix
I	24 h	Negative control	158.3	0.90
		Solvent control^1	100	1.70
		Positive control^6	49.0	8.40S
		Positive control^7	41.0	5.15S
		6.3	159.4	1.55
		12.5	113.2	1.55
		25.0 P	57.4	0.85
Exposure period 4 hrs with S9 mix
IIA	48 h	Negative control	100.8	0.95
		Solvent control^1	100	0.55
		Positive control^8	67.7	15.55S
		Positive control^9	19.9	44.70S
		3.1	82.2	0.70
		6.3	80.9	1.00
		12.5	42.4	0.60

P         precipitation occurred

S        number of micronucleated cells statistically significant higher than corresponding control values

1        deioinised water 10%(v/v)

2         colcemid 7.5µg/mL

3         colcemid 10.0 µg/mL

4         mitomycin C 0.03 µg/mL

5         mitomycin C 0.1 µg/mL

6         cyclophosphamide 10 µg/mL

7         cyclophosphamide 25 µg/mL

8         cyclophosphamide 2.5 µg/mL

9         cyclophosphamide 5.0 µg/mL

Conclusions:

Based on the results of the read across study, the test substance is not expected to induce micronuclei in V79 cells (Chinese hamster cell line), in the absence and the presence of metabolic activation; therefore the substance can be considered to be non-clastogenic.

Executive summary:

An in vitro study was conducted to determine the induction of micronucleus by the read across substance, C22 TMAC (active: 78.8%), using Chinese hamster V79 cells, according to the OECD Guideline 487 (micronucleus test), in compliance with GLP. The read across substance, suspended (Experiment I) or dissolved (Experiments IIA and IIB) in deionised water, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence (exposure for 4 h in Exp I and 20 h in Exp IIA and IIB) and presence (exposure for 4 h) of metabolic activation by S9 mix. In each experimental group, two parallel cultures were analysed and 1000 cells per culture were scored for micronuclei. The following read across substance concentrations were applied (or evaluated):

Exp I: 6.3, 12.5 and 25.0 µg/ml, with S9 mix, 4 h exposure, 24 h preparation interval and 3.1, 6.3 and 12.5 µg/ml, without S9 mix, 4 h exposure, 24 h preparation interval;

Exp IIA: 3.1, 6.3 and 12.5 µg/ml, with with S9 mix (4 h exposure, 48 h preparation interval) and without S9 mix (20 h exposure, 24 h preparation interval);

Exp IIB: 5.0, 6.0, and 7.0 µg/ml, without S9 mix, 20 h exposure, 24 h preparation interval;

In Experiment I, read across substance precipitation was observed at a concentration of 50.0 µg/mL and above in the absence of S9 mix and at 25.0 µg/mL and above in the presence of S9 mix. Clear toxic effects indicated by reduced cell numbers were observed after treatment with 12.5 µg/mL (11.3% of control) and above in the absence of S9 mix. In the presence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In the absence and presence of S9 mix, no biologically relevant increase in the percentage of micronucleated cells was observed up to the highest scorable concentration. The rates of micronucleated cells after treatment with the read across substance in the absence of S9 mix (0.95 - 1.55%) and in the presence of S9 mix (0.85 - 1.55%) were below the corresponding solvent control values (2.00 and 1.70%, respectively) and within the range the laboratory's historical control data: 0.0 - 2.0 % micronucleated cells.

In Experiment IIA, read across substance precipitation was observed at a concentration of 6.3 µg/mL and above in the absence of S9 mix and at 25.0 µg/mL and above in the presence of S9 mix. Clear cytototoxicity (indicated by reduced cell numbers when compared to control values) of about 60% or above were observed after treatment with 12.5 µg/mL (70% cytotoxicity) and above in the absence of S9 mix, and with 12.5 µg/mL (58% cytotoxicity) and above in the presence of S9 mix. In the presence of S9 mix, no biologically relevant increase in the percentage of micronucleated cells was observed up to the highest evaluated concentration of 12.5 µg/mL. The rates of micronucleated cells after treatment with the read across substance (0.60 - 1.00%) were close to the corresponding solvent control value (0.55 %) and within the range of our historical control data: 0.0 - 2.0 % micronucleated cells. In the absence of S9 mix, dose-related increases in the percentage of micronucleated cells (1-65%, 1.75 %, and 2.40%) were observed at the three evaluated concentrations (3.1, 6.3, and 12.5 µg/mL, respectively). Although all three values were statistically significantly increased compared to the corresponding solvent control value (0.85%), the percentages of micronucleated cells at 3.1 and 6.3 µg/mL (1.65% and 1.75%, respectively) were within the laboratorie's historical control data range (0.0 - 2.0% micronucleated cells), and these increases were regarded as biologically irrelevant. At the highest concentration evaluated (12.5µg/mL), given the high cytotoxicity observed (70%), the biological relevance of the increased incidence of micronucleated cells remained ambiguous.

To verify the observation in Experiment IIA, a repeat experiment in the absence of S9 mix within a narrow concentration range, designated Experiment IIB, was performed. The first repeat experiment could not be evaluated, since only two concentrations were available for cytogenetic evaluation, which was not in compliance with the guideline recommendations. In both experiments, concentrations of 7.5 µg/mL and 8.0 µg/mL and above were not scorable for cytogenetic damage due to strong read across substance induced cytotoxicity. In the second repeat experiment, read across substance precipitation was observed at a concentration of 6.0 µglmL and above in the absence of S9 mix. Clear toxic effects indicated by reduced cell numbers were observed after treatment with 7.0 µg/mL (62 % cytotoxicity). No biologically relevant increase in the percentage of micronucleated cells was observed up to the highest scorable concentration. The rates of micronucleated cells after treatment with the read across substance (0.05 - 1.05 %) were close to the corresponding solvent control values (0.55 %) and within the range of our historical control data: 0.0 - 2.0 % micronucleated cells. Accordingly, the increased incidence of micronucleated cells observed at a concentration associated with high cytotoxicity (Experiment IIA in the absence of S9 mix) could not be reproduced, and the finding was considered to bear no biological significance. Colcemid (25 ng/mL to 10 µg/mL), Mitomycin C (0.03 or 0.1 µg/mL) or CPA (2.5 to 25 µg/mL) were evaluated as positive controls and showed a distinct increase in the percentage of micronucleated cells; hence the experiment was considered valid. Under the study conditions, the read across substance did not induce micronuclei in V79 cells (Chinese hamster cell line), in the absence and the presence of metabolic activation; therefore the substance was considered to be non-clastogenic (Hopker, 2007). Based on the results of the read across study, similar absence of clastogenic response is expected for the test substance, C18-22 TMAC in an micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Study 1: An in vitro study was conducted to determine the mutagenic potential of the read across substance, C20-22 TMAC (active content not specified), using bacterial reverser mutation assay (Ames test), according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA 98, TA100, TA 1535 and TA 1537 was used in this experiment. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, i.e., the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the read across substance. The plates were inverted and incubated for 48 h at 37±2°C in dark, colonies were counted. The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. The read across substance was dissolved in ethanol and 6 test concentrations ranging from 4 μg /plate to 5000 μg/plate (i.e., 0, 4, 20, 100, 500, 2500 and 5000 μg per plate) was used. The number of spontaneous revertant colonies in the control plates (without the mutagen) was similar to that described in the literature.Toxicity was observed at 2500 μg/plate and above in the bacterial strains; therefore 5000 μg/plate was chosen as top dose level for the mutagenicity study. The read across substance did not show a dose dependent increase in the number of revertants in any of the bacterial strains in the presence or absence of metabolic activation system. The negative and positive controls gave results within the expected range; hence the experiment was considered valid. Under the study conditions, the read across substance was determined to be non-mutagenic in Ames test, with or without metabolic activation (Muller, 1995). Based on the results of the read across study, similar absence of mutagenic potential is expected for the test substance, C18-22 TMAC, in an Ames test.

Study 2: An in vitro study was conducted to investigate the potential of the read across substance, C22 TMAC (active: 78.8%), to induce gene mutations in V79 Chinese hamster cells, according to OECD Guideline 476, in compliance with GLP. The target for gene mutation was the gene coding for hypoxanthine-guanine phosphoribosyl transferase (HPRT), an enzyme that can convert 6-thioguanine (6-TG) to its toxic ribophosphorylated derivative. Chinese Hamster V79 cells were exposed to the read across substance over a period of either 4 or 24 h (for the 4 hours exposure, with and without metabolic activation by rat liver S9 mix). After exposure, cells were maintained for 7 d read across substance-free under normal growth conditions to deplete HPRT in mutated cells. After this period, cells were maintained for 8 d in 6-TG containing medium, i.e., toxic to non-mutated (HPRT-expressing) cells. After this selection period, the number of colonies remaining (each representing a mutated cell) was counted and their number was compared to those achieved in the solvent controls and positive control substances, known to be mutagenic. Relevant toxic effects indicated by a relative cloning efficiency I (CE I) below 50% occurred in the first experiment at 1.3 μg/mL and above without metabolic activation and at 9.9 (culture I only) and 19.7 μg/mL and above in the presence of metabolic activation. In the second experiment such toxic effects occurred at 4.7 μg/mL test concentration and above. No relevant and reproducible increase of the mutation frequency occurred at any test concentration with and without metabolic activation. All positive controls showed a distinct increase in the number of mutant colonies. All mutant frequencies remained well within the historical range of negative and solvent controls; hence the experiment was considered valid. Under the study conditions, the read across substance was not found to induce gene mutations in the HPRT locus in V79 Chinese hamster cells, either in the presence or absence of metabolic activation (Wollny, 2007).Based on the results of the read across study, a similar absence of mutagenic potential is expected for the test substance, C18-22 TMAC in the HPRT assay.

Study 3: An in vitro study was conducted to determine the induction of micronucleus by the read across substance, C22 TMAC (active: 78.8%), using Chinese hamster V79 cells, according to the OECD Guideline 487 (micronucleus test), in compliance with GLP.The read across substance, suspended (Experiment I) or dissolved (Experiments IIA and IIB) in deionised water, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence (exposure for 4 h in Exp I and 20 h in Exp IIA and IIB) and presence (exposure for 4 h) of metabolic activation by S9 mix. In each experimental group, two parallel cultures were analysed and 1000 cells per culture were scored for micronuclei. The following read across substance concentrations were applied (or evaluated):

Exp I: 6.3, 12.5 and 25.0 µg/ml, with S9 mix, 4 h exposure, 24 h preparation interval and 3.1, 6.3 and 12.5 µg/ml, without S9 mix, 4 h exposure, 24 h preparation interval;

Exp IIA: 3.1, 6.3 and 12.5 µg/ml, with S9 mix (4 h exposure, 48 h preparation interval) and without S9 mix (20 h exposure, 24 h preparation interval);

Exp IIB: 5.0, 6.0, and 7.0 µg/ml, without S9 mix, 20 h exposure, 24 h preparation interval;

In Experiment I, read across substance precipitation was observed at a concentration of 50.0 µg/mL and above in the absence of S9 mix and at 25.0 µg/mL and above in the presence of S9 mix. Clear toxic effects indicated by reduced cell numbers were observed after treatment with 12.5 µg/mL (11.3% of control) and above in the absence of S9 mix. In the presence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In the absence and presence of S9 mix, no biologically relevant increase in the percentage of micronucleated cells was observed up to the highest scorable concentration. The rates of micronucleated cells after treatment with the read across substance in the absence of S9 mix (0.95 - 1.55%) and in the presence of S9 mix (0.85 - 1.55%) were below the corresponding solvent control values (2.00 and 1.70%, respectively) and within the range the laboratory's historical control data: 0.0 - 2.0 % micronucleated cells.

In Experiment IIA, read across substance precipitation was observed at a concentration of 6.3 µg/mL and above in the absence of S9 mix and at 25.0 µg/mL and above in the presence of S9 mix. Clear cytototoxicity (indicated by reduced cell numbers when compared to control values) of about 60% or above were observed after treatment with 12.5 µg/mL (70% cytotoxicity) and above in the absence of S9 mix, and with 12.5 µg/mL (58% cytotoxicity) and above in the presence of S9 mix. In the presence of S9 mix, no biologically relevant increase in the percentage of micronucleated cells was observed up to the highest evaluated concentration of 12.5 µg/mL. The rates of micronucleated cells after treatment with the read across substance (0.60 - 1.00%) were close to the corresponding solvent control value (0.55 %) and within the range of our historical control data: 0.0 - 2.0 % micronucleated cells. In the absence of S9 mix, dose-related increases in the percentage of micronucleated cells (1-65%, 1.75 %, and 2.40%) were observed at the three evaluated concentrations (3.1, 6.3, and 12.5 µg/mL, respectively). Although all three values were statistically significantly increased compared to the corresponding solvent control value (0.85%), the percentages of micronucleated cells at 3.1 and 6.3 µg/mL (1.65% and 1.75%, respectively) were within the laboratorie's historical control data range (0.0 - 2.0% micronucleated cells), and these increases were regarded as biologically irrelevant. At the highest concentration evaluated (12.5µg/mL), given the high cytotoxicity observed (70%), the biological relevance of the increased incidence of micronucleated cells remained ambiguous.

To verify the observation in Experiment IIA, a repeat experiment in the absence of S9 mix within a narrow concentration range, designated Experiment IIB, was performed. The first repeat experiment could not be evaluated, since only two concentrations were available for cytogenetic evaluation, which was not in compliance with the guideline recommendations. In both experiments, concentrations of 7.5 µg/mL and 8.0 µg/mL and above were not scorable for cytogenetic damage due to strong read across substance induced cytotoxicity. In the second repeat experiment, read across substance precipitation was observed at a concentration of 6.0 µglmL and above in the absence of S9 mix. Clear toxic effects indicated by reduced cell numbers were observed after treatment with 7.0 µg/mL (62 % cytotoxicity). No biologically relevant increase in the percentage of micronucleated cells was observed up to the highest scorable concentration. The rates of micronucleated cells after treatment with the read across substance (0.05 - 1.05 %) were close to the corresponding solvent control values (0.55 %) and within the range of our historical control data: 0.0 - 2.0 % micronucleated cells. Accordingly, the increased incidence of micronucleated cells observed at a concentration associated with high cytotoxicity (Experiment IIA in the absence of S9 mix) could not be reproduced, and the finding was considered to bear no biological significance. Colcemid (25 ng/mL to 10 µg/mL), Mitomycin C (0.03 or 0.1 µg/mL) or CPA (2.5 to 25 µg/mL) were evaluated as positive controls and showed a distinct increase in the percentage of micronucleated cells; hence the experiment was considered valid. Under the study conditions, the read across substance did not induce micronuclei in V79 cells (Chinese hamster cell line), in the absence and the presence of metabolic activation; therefore the substance was considered to be non-clastogenic (Hopker, 2007). Based on the results of the read across study, similar absence of clastogenic response is expected for the test substance, C18-22 TMACin an micronucleus assay.

Overall, based on the results of the read across studies, the test substance C18-22 TMAC is considered as non-genotoxic.

Justification for classification or non-classification

Based on the absence of genotoxicity in the read across in vitro studies, the test substance, C18 -22 TMAC does not warrant classification for genotoxicity, according to the EU CLP criteria (Regulation 1272/2008/EC).