Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxicity of the registration substance was investigated in three in-vitro test system.

Negative results were obtained in Ames test, in mammalian gene mutation test and in micronucleus test.

Overall, no significant genotoxicity can be assigned for the registration substance.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 487, Draft Version 1

Deviations:

no

Principles of method if other than guideline:

OECD Guideline Draft Proposal for a new Guideline No. 487, Version 1

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

Remarks:

and colcemid and cyclophosphamide

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 20 hours without S9 mix and 4 hours with metabolic activation. The cells were prepared 24 hours (48 hours for Exp. II with S9 mix) after start of treatment with the test item.

METHOD OF APPLICATION: in minimal essential medium

DURATION
- Exposure duration: 4 and 24 hours
- Expression time (cells in growth medium): 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours (48 hours 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 item 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 item 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/or
- 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.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see textbelow

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item Behentrimonium chloride, 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 hours 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 hours with metabolic activation. The cells were prepared 24 hours (Exp. I, IIA and llB) and 48 hrs (Exp. IIA) after start of treatment with the test item.
In each experimental group two parallel cultures were set up. Per culture 1000 cells were scored for micronuclei.
Evaluated experimental points after treatment with Behentrimonium chloride:
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 item 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 item 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 item 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 item (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 item induced cytotoxicity.
In the second repeat experiment, test item 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 item (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 item 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 item Behentrimonium chloride did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and the presence of metabolic activation.
Therefore, Behentrimonium chloride has to be considered as non-mutagenic in this in vitro test system, when tested up to cytotoxic test item 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

Remarks:

Migrated from field 'Test system'. Remarks: V79

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 item, 5000.0 µg/mL of Behentrimonium chloride (approx. 9.7 mM) were applied as top concentration in Experiment I. Test item 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 item concentrations of 9.8 µg/mL and above in the absence and presence of metabolic activation, Experiment I was repeated with test item concentrations between 0.8 and 400.0 µg/mL (with and without S9 mix). With regard to the purity of the test item, the corresponding amount of the active substance in the respective test item concentrations was 78.8 %.

In Experiment I, precipitation of the test item 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 item 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 item concentrations between 0.5 and 15.0 µg/mL.

Summary of results of the micronucleus test with Behentrimonium chloride

Exp.	Preparation	Test item	Cell number	Micronucleated
	interval	concentration	In %	cells
		in µg/mL	of control	in %
Exposure period 4 hrs without S9 mix
I	24 hrs	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 hrs	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 hrs	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 hrs	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 hrs	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:

The genotoxicity of the registration substance was investigated according to the guideline OECD 487. Negative result was obtained.

Executive summary:

The registration substance was assessed for its clastogenicity according to the guideline OECD 487.

The registration substance did not induce micronuclei in V79 cells (Chinese hamster cell line) in the absence and the presence of metabolic activation at up to the concentration associated with clear cytoxicity. No significant clastogenicity was found.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-07 - 2007-03

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

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:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

other: see text below

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 item 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 item 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.

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 item. The FCS-concentration is 10 % during long term treatment compared to 0 % during 4 h treatment. Test items 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 item 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 hour 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 item -- 0.1 -- 92.8 -- culture was not continued

Test item -- 0.2 -- 100.8 -- 103.0 -- 2.0 -- 0.3

Test item -- 0.3 -- 99.5 -- 92.4 -- 5.6 -- 0.9

Test item -- 0.6 -- 98.3 -- 80.6 -- 12.4 -- 2.0

Test item --1.3 -- 22.7 -- 80.6 -- 2.1 -- 0.3

Test item -- 1.9 -- 0.0 -- 34.9 -- 3.4 -- 0.5

Exp. I, 4 hour 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 item -- 0.1 -- 85.9 -- culture was not continued

Test item -- 0.2 -- 87.9 -- 111.0 -- 1.5 -- 0.5

Test item -- 0.3 -- 85.8 -- 122.9 -- 3.7 -- 1.3

Test item -- 0.6 -- 76.8 -- 113.4 -- 7.3 -- 2.5

Test item --1.3 -- 21.5 -- 78.1 -- 4.8 -- 1.6

Test item -- 1.9 -- 0.0 -- 30.2 -- 1.5 -- 0.5

Exp. I, 4 hour 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 item -- 1.3 -- 87.2 -- 77.3 -- 1.2 -- 0.3

Test item -- 2.4 -- 82.5 -- 78.4 -- 1.4 -- 0.4

Test item -- 5.0 -- 75.5 -- 58.6 -- 7.9 -- 2.2

Test item -- 9.9 -- 3.6 -- 34.5 -- 4.4 -- 1.2

Test item --19.7 -- 0.0 -- 20.0 -- 3.6 -- 1.0

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

Exp. I, 4 hour 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 item -- 1.3 -- 97.9 -- 96.8 -- 7.8 -- 0.8

Test item -- 2.4 -- 104.6 -- 102.9 -- 7.8 -- 0.8

Test item -- 5.0 -- 93.4 -- 61.6 -- 1.4 -- 0.1

Test item -- 9.9 -- 78.5 -- 58.0 -- 4.6 -- 0.5

Test item -- 19.7 -- 5.1 -- 26.5 -- 3.7 -- 0.4

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

Exp.II, 24 hour 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 item -- 0.6 -- 98.1 -- 96.6 -- 7.4 -- 2.3

Test item -- 1.2 -- 91.5 -- 78.0 -- 4.6 -- 1.4

Test item -- 2.4 -- 64.7 -- 30.3 -- 6.9 -- 2.1

Test item -- 4.7 -- 0.0 -- 6.5 -- 2.7 -- 0.8

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

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

Exp.II, 24 hour 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 item -- 0.6 -- 104.3 -- 75.3 -- 6.8 -- 1.3

Test item -- 1.2 -- 94.4 -- 73.5 -- 4.2 -- 0.8

Test item -- 2.4 -- 74.3 -- 37.5 -- 8.3 -- 1.6

Test item -- 4.7 -- 0.0 -- 9.8 -- 7.8 -- 1.5

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

Test item -- 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:

The genotoxicity of the registration substance was investigated according to the guideline OECD 476. Negative result was obtained.

Executive summary:

The genotoxic potential of the registration substance was evaluated in a mammalian cell gene mutation assay according to OECD guideline 476.

No increase of the mutation frequency occurred at up to concentrations associated with clear cytotoxicity with and without metabolic activation. No mutagenicity was found.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 May 2020 - 21 Aug 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

The S9 liver microsomal fraction was obtained from Trinova Biochem GmbH, Gießen, Germany. Male Sprague Dawley rats were induced with phenobarbital/β-naphthoflavone.
The following quality control determinations were performed by Trinova Biochem GmbH:
a) Alkoxyresorufin-O-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene)
A stock of the supernatant containing the microsomes was frozen in aliquots of 5 mL and stored at  -75 °C.
The protein concentration in the S9 preparation was 37 mg/mL (Lot: 4222) and was adjusted to 30 mg/mL.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other:

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

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

Untreated negative 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

Untreated negative 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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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

Conclusions:

The genotoxicity of the registration substance was investigated according to the guideline OECD 471. Negative result was obtained.

Executive summary:

The registration substance was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and with test strain E. coli WP2 uvrA (pkm101).

The registration substance did not increase the number of revertants in any of the bacterial strains with and without metabolic activation. No significant mutagenicity was found.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Due to the deficiency of the available Ames test (E-coli strain missing; no pre-incubation method as the second experiment) a new study was initiated in May 2020. The result will be available in July 2020.

Justification for classification or non-classification

The genotoxicity of the registration substance was investigated in three in-vitro test system.

Negative results were obtained in Ames test, in mammalian gene mutation test and in micronucleus test. No classification is warranted.