Quaternary ammonium compounds, benzyl-C16-18-alkyldimethyl, chlorides

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results from an in vitro genotoxicity battery with the test as well as read across substance, C16 -18 ADBAC is not considered to have any genotoxic potential.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 23 January, 2012 to 02 Feburary, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without S9-mix: 0.1, 0.3, 1, 3, 10 and 33 µg/plate
With S9-mix: 0.3, 1, 3, 10, 33 and 66 µg/plate
Experiment 2:
TA1535, TA1537 and TA100
Without S9-mix: 0,1, 0,3, 1, 3, 10 and 33 µg/plate
With S9-mix: 0.3, 1, 3, 10, 33 and 66 µg/plate
TA98
Without S9-mix: 1, 3, 10, 33, 66 and 100 µg/plate
With S9-mix: 0.3, 1, 3, 10, 33 and 66 µg/plate
WP2uvrA
Without S9-mix: 1, 3, 10, 33, 66 and 100 µg/plate
With S9-mix: 1, 3, 10, 33, 100 and 333 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 Migrated to IUCLID6: 650 µg/plate in DMSO for TA100

Positive control substance:

2-nitrofluorene

Remarks:

without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA

Positive control substance:

sodium azide

Remarks:

without S9 Migrated to IUCLID6: 5 µg/plate in saline for TA1535

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9: 10 µg/plate and above and with S9: 33 µg/plate and above

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:

without S9: 3 µg/plate and above and with S9: 10 µg/plate and above

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:

without S9: 100 µg/plate and above and with S9: 10 µg/plate and above

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:

without S9: 10 µg/plate and above and with S9: 33 µg/plate and above

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9: 66 µg/plate and above and with S9: 333 µg/plate and above

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 10 μg/plate and above in the absence of S9-mix and at 33 µg/plate in the presence of S9-mix. In tester strain WP2uvrA, toxicity was observed at dose levels of 100 μg/plate and above in the absence of S9-mix and at 333 µg/plate in the presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without S9: 10 µg/plate and above and with S9: 33 µg/plate and above
TA1537: without S9: 3 µg/plate and above and with S9: 10 µg/plate and above
TA98: without S9: 100 µg/plate and above and with S9: 10 µg/plate and above
TA100: without S9: 10 µg/plate and above and with S9: 33 µg/plate and above
WP2uvrA: without S9: 66 µg/plate and above and with S9: 333 µg/plate and above

Conclusions:

Under the study conditions, the test substance was not mutagenic in S. typhimurium strains TA1535, TA1537, TA98, TA100 and in the Escherichia coli strain WP2 uvr A,with and without metabolic activation.

Executive summary:

An in vitro study was conducted to determine the mutagenic potential of the test substance, C16-18 ADBAC (98.2% active), in S. typhimurium strains TA1535, TA1537, TA98, TA100 and in the Escherichia coli strain WP2 uvr A, according to OECD 471 Guideline and EU Method B.13/14, in compliance with GLP. Six concentration levels of the test substance for each bacterial strain were tested in triplicate with and without a metabolic activation system (S9). The concentration-range was determined in a preliminary toxicity assay and was 0.1 to 66 µg/plate in the first experiment. The experiment was repeated on a separate day using the same concentration-range, fresh cultures of the bacterial strains and fresh test substance formulations. The test substance did not induce a significant concentration-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9. These results were confirmed in an independent repeat. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, the test substance was not mutagenic in S. typhimurium strains TA1535, TA1537, TA98, TA100 and in the Escherichia coli strain WP2 uvr A,with and without metabolic activation (Verspeek-Rip, 2012).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Study period:

From March 22, 2001 to September 25, 2001

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.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - 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: human

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone and ß-naphthoflavone - induced rat liver S9 fraction

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 19.5, 39, 78.1, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL (with and without activation)
Chromosome aberration test:
Experiment 1: 0, 4, 8, 16, 20 µg/mL (with and without activation)
Experiment 2: 0, 4, 8, 12, 16, 24 µg/mL (with and without activation)

Vehicle / solvent:

Eagle’s minimal essential medium with HEPES buffer (MEM)

Untreated negative controls:

yes

Remarks:

Negative (media) control

Negative solvent / vehicle controls:

yes

Remarks:

Eagle’s minimal essential medium with HEPES buffer (MEM)

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

(Without S9, at 0.4 and 0.25 µg/mL in Experiment 1 and 2 respectively)

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

(With S9, at 12.5 and 10.0 µg/mL in experiment 1 and 2 respectively)

Details on test system and experimental conditions:

Method of application: In Eagle’s minimal essential medium with HEPES buffer (MEM)
Duration:
- Exposure duration:
Without metabolic activation: 4 and 24h
With metabolic activation: 4h
- Exposure procedure: The cultures were incubated at 37˚C for 4 or 24h (as appropriate) in the presence of the test substance at predetermined concentrations/vehicle/positive controls with or without the S9 reaction mixture.
- Expression time: Approximately 20h after initiation of treatment
- Fixation time: 4h
Spindle inhibitor: Demecolcine (colcemid, 0.1 μg/mL) was added approx. 2 h prior to harvest timeSpindle
Stain: When the slides were dry they were stained in 5% Gurrs Giemsa solution for 5 minutes, rinsed, dried and coverslipped using mounting medium.
Number of replications: At least 2 slides/ flask
Number of cell evaluated: 100 consecutive well-spread metaphase cells (if possible), from each culture were counted, and if the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted.
Determination of the cytotoxicity: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Statistics:

The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells were compared, where necessary, with the concurrent vehicle control value using Fisher’s Exact test and chi-squared test.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slightly toxic at 20 µg/mL in experiment 1 (with S9 activation) and toxic at 16 µg/mL in experiment 1 and at 20 µg/mL in experiment 2 (without S9 activation))

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Results of chromosomal aberration in human lymphocytes (Experiment 1)

Treatment (μg/mL)	S9 Activation	Treatment Time	Mean Mitotic Index	Cells Scored	Total Number of Aberrations +Gaps - Gaps	Cells with Numerical Aberrations + Gaps(%)	Cells with Structural Aberrations - Gaps(%)
Vehicle	-	4	4.2	200	11    7	5.0	3.5
Test substance
4	-	4	3.2	200	6   1	3.0	0.5
8	-	4	2.7	200	2   1	1.0	0.5
16	-	4	1.5	200	7   5	3.0	2.0
Positive control (MMC) 0.4	-	4	2.2	200	83   64	32.0	26.0**
Vehicle	+	4	3.9	200	5    1	2.5	0.5
Test substance
8	+	4	2.3	200	4   1	2.0	0.5
16	+	4	2.1	200	7   4	2.5	1.0
20	+	4	1.6	200	7   3	3.5	1.5
Positive control (CP) 12.5	+	4	1.4	300	76   45	20.0	12.0**

Treatment: Cells from the 4-h treatment regimens were harvested 20h after the initiation of the treatments.

                         Frequency of Aberrant Cells: **, p ≤ 0.001

Table 2. Results of chromosomal aberration in human lymphocytes (Experiment 2)

Treatment (μg/mL)	S9 Activation	Treatment Time	Mean Mitotic Index	Cells Scored	Total Number of Aberrations +Gaps - Gaps	Cells with Numerical Aberrations + Gaps(%)	Cells with Structural Aberrations - Gaps(%)
Vehicle	-	4	7.0	200	4    1	2.0	0.5
Test substance
4	-	24	4.9	200	6   4	2.0	1.0
8	-	24	2.7	200	6   6	2.5	2.5
12	-	24	2.6	200	12   2	6.0	1.0
Positive control (MMC) 0.2	-	24	2.3	200	115   86	37.0	30.0**
Vehicle	+	4	5.9	200	8    4	2.5	0.5
Test substance
8	+	4	5.2	200	7   3	3.5	1.5
16	+	4	3.3	200	0   0	0	0
24	+	4	3.4	200	1   1	0.5	0.5
Positive control (CP) 12.5	+	4	1.4	200	108   79	33.5	27.0**

Treatment: Cells from both the 4-h and 24 h treatment regimens were harvested 20 h after the initiation of the treatments.

Frequency of Aberrant Cells: **, p ≤ 0.001

For further details, refer to the attachment under 'Attached background material'

Conclusions:

Based on the results of the study, the read across substance is not considered to be non-clastogenic to human lymphocytes with and without metabolic activation.

Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the test substance, C12-16 ADBAC (49.7% active in water) in chromosome aberration test, according to OECD Guideline 473 and EU Method B.10, in compliance with GLP. This experiment was performed in human lymphocyte cells. Duplicate cell cultures of human lymphocytes, treated with the test substance, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls (mitomycin-C (without S9) and cyclophosphamide (with S9)). Four treatment conditions were used for the study. Experiment 1 and 4 h exposure with and without metabolic activation was followed by a 20 h expression period. In Experiment 2, the 4 h exposure with metabolic activation was repeated while in the absence of metabolic activation the exposure time was increased to 24 h. The doses studied were 0, 4, 8, 16, 20 µg/mL (with and without activation) in Experiment 1 and 0, 4, 8, 12, 16, 24 µg/mL (with and without activation) in Experiment 2. The test substance was considered negative for chromosomal aberrations in human lymphocytes in vitro under the S9 metabolic activation and non-activation conditions of the assay. There was no indication of chromosomal ploidy changes in cultures exposed to the test substance in either the presence or absence of S9 mix. Mutant frequencies of all cultures treated with the test substance were within the acceptable range for background mutant frequencies. Based on the results of the study, the test substance is not considered to be non-clastogenic to human lymphocytes with and without metabolic activation (Durward, 2001). Based on the results of the read across study, a similar non-genotoxic potential can be expected for the test substance.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Refer to the 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 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

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

Details on test system and experimental conditions:

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

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

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

Conclusions:

Under the study conditions, the read across substance was not clastogenic in Chinese hamster ovary cells with or without metabolic activation.

Executive summary:

An in vitro study was conducted to determine the clastogenic potential of the read across substance, C12-16 ADBAC (50% active in water) according to OECD Guideline 473 (chromosome aberration test), in compliance with GLP. This experiment was realized in Chinese hamster ovary cells. Based on preliminary cytotoxicity test, 0, 0.36, 1.09, 3.27 and 9.80 µg/mL read across substance (without S9) as well as 0, 6.13, 12.25, 24.5, 49.0 and 98.0 µg/mL (with metabolic activation of S9 from rats treated with Aroclor 1254) read across substance were selected. The exposure times were 21h for the experiments without metabolic activation, and 3h for the experiments with metabolic activation, with harvesting times of 12 and 21h. In the preliminary toxicity test, in the absence of S9-mix, the substance was toxic at a concentration of 15.68 µg/mL and above; and in the presence of S9-mix, the substance was very toxic at 78.4 µg/mL and above at both harvesting times. The read across substance did not induce a statistically significant increase in the number of cells with structural chromosome aberrations (breaks, exchanges and multiple aberrations) at any of the concentrations used, either in the absence or in the presence of the S-9 mix. Under the study conditions, the read across substance was not clastogenic in Chinese hamster ovary cells with or without metabolic activation (Willmer, 1986). Based on the results of the read across study, a similar non-genotoxic potential can be expected for the test substance.

Reference 4

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 May 11, 1987 to September 28, 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to 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.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: U.S. EPA FIFRA 84-4

Version / remarks:

1989

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Cell Type: CHO-K1-BH4

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9 fraction

Test concentrations with justification for top dose:

Preliminary cytotoxicity assay: 1, 2, 5, 10, 20, 50, 100, 200, 500 and 5000 µg/mL (With and without metabolic activation)

Mutation assay (Without S-9)
Trial I: 1, 5, 10, 13, 16, 20, 25, 35, 50, and 65 µg/mL
Trial II: 1, 5, 10, 12, 14, 16, 18, 20, and 24 µg/mL

Mutation assay (With S-9)
Trial I: 1, 5, 10, 20, 30, 40, 50, 65, 85 and 100 µg/mL
Trial II: 10, 20, 22, 24, 26, 28, 30, 40 and 50 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

(Negative (media) controls)

Negative solvent / vehicle controls:

yes

Remarks:

(10% water in culture medium)

Positive controls:

yes

Positive control substance:

other: 5-Bromo-2’-deoxyuridine (BrdU)

Remarks:

(without metabolic activation)

Positive controls:

yes

Positive control substance:

other: 3-Methycholanthrene (MCA)

Remarks:

(with metabolic activation)

Details on test system and experimental conditions:

Method of applicfation: Single monolayer culture of CHO cells (in medium).

Duration:
- Exposure duration: 4h (with and without metabolic activation)
- Exposure procedure: The cells were quantitatively seeded at 200 cells/dish (cytotoxicity assay) and at about 3 X 10 (6) cells/T-75 (75 cm2) tissue culture flask on the day before dosing. After18h, cell cultures were exposed to test or control substances for 4h.
- Expression time: Approximately 7d after initiation of treatment
- Fixation time: 7d after treatment initiation.

Stain: Colonies were fixed in alcohol, stained with Giemsa solution and counted by eye, excluding those with approximately 50 cells or less.

Number of cells evaluated: 200 cells/dish

Determination of cytotoxicity
- Method: Following parameters were calculated in the study:
1) Relative survival to treatment: The average number of colonies in three dishes (seeded at 200 cells each) was determined.
Relative survival (%) = (Average number of colonies per treated culture/average number of colonies per vehicle control dish) X 100%
2) Relative population growth: This parameter showed the cumulative growth of the treated cell population, relative to the vehicle control growth, over the entire expression period and prior to mutant selection.
Relative population growth (%) = (Treated culture population increase over the expression period/vehicle control population increase over the expression period) X 100%
3) Absolute cloning efficiency: The ability of the cells to form colonies at the time of mutant selection is measured by the absolute cloning efficiency (CE).
Absolute CE (%) = [Average number of viable colonies per dish/200] x 100%
4) Mutant frequency: The mutant frequency is calculated as the ratio of colonies found in thioguanine-selection medium to the total number of cells seeded, adjusted by the absolute CE.
Mutant Frequency = Total mutant clones/[number of dishes X 2x10(5) X absolute CE]

Evaluation criteria:

Evaluation of a positive response:
To evaluate the test substance as a mutagen following criterion should be met:
- The mutant frequency must meet or exceed 15 x 10E-6 in order to compensate for random fluctuations in the 0 to 10E-6 background mutant frequencies that are typical for this assay.
- A dose related or toxicity related increase in mutant frequency for at least 3 doses should be observed.
- If an increase in mutant frequency is observed for a single dose near the highest testable toxicity, as defined previously and the number of mutant colonies is more than twice the value needed to indicate a significant response, the test substance generally will be considered mutagenic.

Evaluation of a Negative Response:
A test substance is evaluated as nonmutagenic in a single assay only if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to concentrations causing about 10 to 15% survival or extends to a concentration at least 75% of that causing excessive toxicity.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

completely toxic at 20 µg/mL and higher concentration (without activation) and at 40 µg/mL and higher concentration (with activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Mutant frequencies of all cultures treated with the test material were within the acceptable range for background mutant frequencies (0 to 15E-06 without S9 mix and 0 to 13.5E-06 with S9 mix).
- Mutation Frequency/10(6) cells for trial I and II (without S9 mix) were as follows:
Trial I:
Control: 0, 7.2
Treated: 0 – 4.0
Positive Control (BrdU): 133.3
Trial II:
Control: 8.9, 2.9
Treated: 0.8 – 14.6 (all within historical control range)
Positive Control (BrdU): 114.0
- Mutation Frequency/10(6) cells for trial I and II (with S9 mix) were as follows:
Trial I:
Control: 6.9, 0
Treated: 0.8 – 6.6
Positive Control (3-MCA): 235.3
Trial II:
Control: 1.3, 2.9
Treated: 0.8 – 6.5 (all within historical control range)
Positive Control (3-MCA): 131.7

For details on results, please refer to the attachment under 'Attached background material'

Conclusions:

Based on the results of the study, the read across substance is not considered to induce any forward mutations at the HGPRT locus in CHO cells with and without metabolic activation.

Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the test substance, C12-16 ADBAC (81.09% active in aqueous/ethanol solution) in a mammalian cell gene mutation test, according to US EPA FIFRA 84-4 or a method similar to US EPA OPPTS 870.5300, in compliance with GLP. The study was performed on the HGPRT locus in Chinese hamster ovary (CHO) cells at test substance concentrations ranging from 0 to100 µg/mL. Preliminary cytotoxicity test showed the test substance to be slightly more toxic without S9 metabolic activation than with activation. The test substance was completely toxic at 20 µg/mL and higher without activation and completely toxic at 40 µg/mL and higher with activation. Dose levels selected for the first trial of the mutation assays covered nontoxic and highly toxic doses. Two independent non-activation and S9 metabolic activation assays were performed. Mutant frequencies of all cultures treated with the test substance were within the acceptable range for background mutant frequencies (0 to 13.5 x 10-6 with S9 mix and 0 to 15 x 10-6 without S9 mix). Based on the results of the study, the test substance is not considered to induce any forward mutations at the HGPRT locus in CHO cells with and without metabolic activation (Young, 1989). Based on the results of the read across study, a similar non-genotoxic potential can be expected for the test substance.

Reference 5

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:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

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

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

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

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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

Conclusions:

Under the study conditions, the read across substance did not show any mutagenic activity at the HGPRT locus in CHO cells with or without metabolic activation.

Executive summary:

An in vitro study was conducted to determine the mutagenic potential of the test substance, C12-16 ADBAC (50% active in water) according to OECD Guideline 476 (cell gene mutation test), in compliance with GLP. This study was performed to evaluate the potential to induce mutations at the HGPRT locus in Chinese hamster ovary (CHO) cells. Based on a preliminary toxicity test, 0, 5, 10, 12.5, 15, 17.5 and 20 nL/mL (without metabolic activation) and 0, 15, 30, 45, 60, 75 and 90 nL/mL (with metabolic activation) were selected for the experiment. The entire experiment was repeated to confirm the results of the first experiment. Five h exposure was used both with and without S9-mix. The test substance did not induce dose-related increase in the mutant frequency at the HGPRT locus at any dose level, either with or without metabolic activation. Exposure to the highest concentrations tested resulted in significant (initial) toxicity for the CHO cells; this indicates that the test was carried out with appropriate concentrations of the test substance. Exposure to positive control substances gave the expected increases in the mutation frequency. The toxicity of the test substance for CHO cells was clearly reduced in the presence of metabolic activation. Under the study conditions, test substance did not show any mutagenic activity at the HGPRT locus in CHO cells with and without metabolic activation (Davis, 1986). Based on the results of the read across study, a similar non-genotoxic potential can be expected for the test substance.

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 test substance, C16-18 ADBAC (98.2% active), in S. typhimurium strains TA1535, TA1537, TA98, TA100 and in the Escherichia coli strain WP2 uvr A, according to OECD 471 Guideline and EU Method B.13/14, in compliance with GLP. Six concentration levels of the test substance for each bacterial strain were tested in triplicate with and without a metabolic activation system (S9). The concentration-range was determined in a preliminary toxicity assay and was 0.1 to 66 µg/plate in the first experiment. The experiment was repeated on a separate day using the same concentration-range, fresh cultures of the bacterial strains and fresh test substance formulations. The test substance did not induce a significant concentration-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9. These results were confirmed in an independent repeat. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, the test substance was not mutagenic in S. typhimurium strains TA1535, TA1537, TA98, TA100 and in the Escherichia coli strain WP2 uvr A,with and without metabolic activation (Verspeek-Rip, 2012).

 

Study 2: An in vitro study was conducted to determine the clastogenic potential of the read across substance, C12-16 ADBAC (49.7% active in water) in chromosome aberration test, according to OECD Guideline 473 and EU Method B.10, in compliance with GLP. This experiment was performed in human lymphocyte cells. Duplicate cell cultures of human lymphocytes, treated with the read across substance, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls (mitomycin-C (without S9) and cyclophosphamide (with S9)). Four treatment conditions were used for the study. Experiment 1 and 4 h exposure with and without metabolic activation was followed by a 20 h expression period. In Experiment 2, the 4 h exposure with metabolic activation was repeated while in the absence of metabolic activation the exposure time was increased to 24 h. The doses studied were 0, 4, 8, 16, 20 µg/mL (with and without activation) in Experiment 1 and 0, 4, 8, 12, 16, 24 µg/mL (with and without activation) in Experiment 2. The read across substance was considered negative for chromosomal aberrations in human lymphocytes in vitro under the S9 metabolic activation and non-activation conditions of the assay. There was no indication of chromosomal ploidy changes in cultures exposed to the read across substance in either the presence or absence of S9 mix. Mutant frequencies of all cultures treated with the read across substance were within the acceptable range for background mutant frequencies. Based on the results of the study, the read across substance is not considered to be non-clastogenic to human lymphocytes with and without metabolic activation (Durward, 2001).

 

Study 3: An in vitro study was conducted to determine the clastogenic potential of the read across substance, C12-16 ADBAC (50% active in water) according to OECD Guideline 473 (chromosome aberration test), in compliance with GLP. This experiment was realized in Chinese hamster ovary cells. Based on preliminary cytotoxicity test, 0, 0.36, 1.09, 3.27 and 9.80 µg/mL read across substance (without S9) as well as 0, 6.13, 12.25, 24.5, 49.0 and 98.0 µg/mL (with metabolic activation of S9 from rats treated with Aroclor 1254) read across substance were selected. The exposure times were 21h for the experiments without metabolic activation, and 3h for the experiments with metabolic activation, with harvesting times of 12 and 21h. In the preliminary toxicity test, in the absence of S9-mix, the substance was toxic at a concentration of 15.68 µg/mL and above; and in the presence of S9-mix, the substance was very toxic at 78.4 µg/mL and above at both harvesting times. The read across substance did not induce a statistically significant increase in the number of cells with structural chromosome aberrations (breaks, exchanges and multiple aberrations) at any of the concentrations used, either in the absence or in the presence of the S-9 mix. Under the study conditions, the read across substance was not clastogenic in Chinese hamster ovary cells with or without metabolic activation (Willmer, 1986).

An in vitro study was conducted to determine the mutagenic potential of the read across substance, C12-16 ADBAC (81.09% active in aqueous/ethanol solution) in a mammalian cell gene mutation test, according to a method similar to US EPA OPPTS 870.5300, in compliance with GLP. The study was performed on the HGPRT locus in Chinese hamster ovary (CHO) cells at read across substance concentrations ranging from 0 to100 µg/mL. Preliminary cytotoxicity test showed the read across substance to be slightly more toxic without S9 metabolic activation than with activation. The read across substance was completely toxic at 20 µg/mL and higher without activation and completely toxic at 40 µg/mL and higher with activation. Dose levels selected for the first trial of the mutation assays covered nontoxic and highly toxic doses. Two independent non-activation and S9 metabolic activation assays were performed. Mutant frequencies of all cultures treated with the read across substance were within the acceptable range for background mutant frequencies (0 to 13.5 x 10-6 with S9 mix and 0 to 15 x 10-6 without S9 mix). Based on the results of the study, the read across substance is not considered to induce any forward mutations at the HGPRT locus in CHO cells with and without metabolic activation (Young, 1989).

 

Study 4: An in vitro study was conducted to determine the mutagenic potential of the read across substance, C12-16 ADBAC (50% active in water) according to OECD Guideline 476 (cell gene mutation test), in compliance with GLP. This study was performed to evaluate the potential to induce mutations at the HGPRT locus in Chinese hamster ovary (CHO) cells. Based on a preliminary toxicity test, 0, 5, 10, 12.5, 15, 17.5 and 20 nL/mL (without metabolic activation) and 0, 15, 30, 45, 60, 75 and 90 nL/mL (with metabolic activation) were selected for the experiment. The entire experiment was repeated to confirm the results of the first experiment. Five h exposure was used both with and without S9-mix. The read across substance did not induce dose-related increase in the mutant frequency at the HGPRT locus at any dose level, either with or without metabolic activation. Exposure to the highest concentrations tested resulted in significant (initial) toxicity for the CHO cells; this indicates that the test was carried out with appropriate concentrations of the read across substance. Exposure to positive control substances gave the expected increases in the mutation frequency. The toxicity of the read across substance for CHO cells was clearly reduced in the presence of metabolic activation. Under the study conditions, read across substance did not show any mutagenic activity at the HGPRT locus in CHO cells with and without metabolic activation (Davis, 1986).

The biocides assessment reports available from RMS Italy on C12-16 ADBAC, also concluded that the read across substance, C12-16 ADBAC, can be considered not genotoxic based onin vitro(Ames test, Chromosomal aberration test, Mammalian cell gene mutation assay) andin vivotest (Chromosomal aberration test in rat bone marrow) (ECHA biocides assessment report, 2015).

Overall, based on the available information from the read across studies, the test substance is not considered to have genotoxic potential. 

Justification for classification or non-classification

Based on the results from an in vitro Ames asay with the test substance as well as in vitro chromosomal aberration and mammalian mutagenicity assays with the read across substance, no classification for genotoxicity is warranted for the test substance according to EU CLP criteria (Regulation EC 1272/2008).