Quaternary ammonium compounds, C12-14-alkyl[(ethylphenyl)methyl]dimethyl, chlorides

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of an in vitro Ames test with the test substance and an in vitro mammalian cytogenicity and mammalian gene mutation assays with read across substance, the test substance, C12 -14 ADEBAC can be considered to be non-genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 21, 2017 to December 18, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Mitochondrial supernatant (S9) prepared from livers of phenobarbital/β-naphthoflavone-induced rats

Test concentrations with justification for top dose:

Preliminary test: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Experiment 1: 0.0316, 0.100, 0.316, 1.0, 3.16, 10.0, 31.6 and 100 µg/plate
Experiment 2: 0.050, 0.158, 0.50, 1.58, 5.00, 15.8, 50.0 and 100 µg/plate

Vehicle / solvent:

Purified water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene and 4-nitro-o-phenylene-diamine

Details on test system and experimental conditions:

Preparation of the Test Substance
The test substance was dissolved in A. dest. and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity. A correction factor of 2.0 was applied to consider the purity of the test substance.

Controls
Solvent / negative as well as positive controls were included in each experiment. Strain specific positive controls were included in the assay, which demonstrated the effective performance of the test.

Negative/Solvent Controls
Negative/solvent controls (A. dest., Eurofins Munich, Lot No. 171107, 171118, 171004) were treated in the same way as all dose groups.

Positive Controls
Without metabolic activation
Tester Strains: S. typhimurium: TA 100, TA 1535
Name: NaN3; sodium azide
CAS No.: 26628-22-8
Supplier: Sigma
Batch No.: STBF8665V
Dissolved in: A. dest.
Concentration: 10 µg/plate
 
Tester Strains: S. typhimurium: TA 98, TA 1537
Name: 4-NOPD; 4-nitro-o-phenylene-diamine
CAS No.: 99-56-9
Supplier: Fluka
Batch No.: MKBQ2637V
Dissolved in: DMSO
Concentrations: 10 µg/plate for TA 98, 40 µg/plate for TA 1537

Tester Strain: S. typhimurium: TA 102
Name: MMS; methylmethanesulfonate
CAS No.: 66-27-3
Supplier: Sigma
Batch No.: MKBX5165V
Dissolved in: A. dest.
Concentration: 1 µL/plate

With metabolic activation
Tester Strains: S. typhimurium: TA 98, TA 100, TA 1535, TA 1537 and TA 102
Name: 2-AA; 2-aminoanthracene
CAS No.: 613-13-8
Supplier: Aldrich
Batch No.: STBD3302V
Dissolved in: DMSO
Concentrations: 2.5 µg/plate; 10 µg/plate for TA 102
The stability of the positive control substances in solution is unknown but a mutagenic response in the expected range is sufficient evidence of biological stability.

Test System: Bacteria
Five strains of S. typhimurium with the following characteristics were used:
TA 98: his D 3052; rfa-; uvrB-; R-factor: frame shift mutations
TA 100: his G 46; rfa-; uvrB-; R-factor: base-pair substitutions
TA 1535: his G 46; rfa-; uvrB-: base-pair substitutions
TA 1537: his C 3076; rfa-; uvrB-: frame shift mutations
TA 102: his G 428 (pAQ1); rfa-; R-factor: base-pair substitutions

Tester strains TA 98, TA 1535 and TA 102 were obtained from MOLTOX, INC., NC 28607, USA. Tester strains TA 100 and TA 1537 were obtained from Xenometrix AG, Switzerland. They were stored as stock cultures in ampoules with nutrient broth (OXOID) supplemented with DMSO (approx. 8% v/v) over liquid nitrogen. All Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. They contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain of the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene coding for a protein of the DNA nucleotide excision repair system resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chl) and biotin (bio) genes (bacteria require biotin for growth). The tester strains TA 98, TA 100 and TA 102 contain the R-factor plasmid, pkM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non R-factor parent strains. pkM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system which is normally present in these organisms. The properties of the S. typhimurium strains with regard to membrane permeability, ampicillin- and tetracycline-resistance as well as normal spontaneous mutation rates are checked regularly according to Ames et al. In this way it is ensured that the experimental conditions set up by Ames are fulfilled.

S9 Homogenate
The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.
The following quality control determinations are performed:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL and stored at ≤75 °C. The protein concentration in the S9 preparation (Lot: 150917, 020617) was 34.0 and 34.2 mg/mL, respectively.

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test substance is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. A test substance producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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

Results

No precipitation of the test substance was observed in any tester strain used in experiment I and II (with and without metabolic activation). Toxic effects of the test substance were noted in all tester strains evaluated in experiment I and II. In experiment I toxic effects of the test substance were observed in tester strain TA 98 at concentrations of 10.0 µg/plate and higher (without metabolic activation) and at concentrations of 31.6 µg/plate (with metabolic activation). In tester strain TA 100 toxic effects of the test substance were noted at concentrations of 31.6 µg/plate and higher (without metabolic activation) and at concentrations of 100 µg/plate (with metabolic activation). In tester strain TA 1535 toxic effects of the test substance were noted at concentrations of 10.0 µg/plate and higher (with and without metabolic activation). In tester strain TA 1537 toxic effects of the test substance were noted at concentrations of 31.6 µg/plate and higher (with and without metabolic activation). In tester strain TA 102 toxic effects of the test substance were observed at concentrations of 10.0 µg/plate and higher (without metabolic activation) and at concentrations of 100 µg/plate and higher (with metabolic activation). In experiment II toxic effects of the test substance were noted in tester strains TA 98 and TA 1535 at concentrations of 15.8 µg/plate and higher (without metabolic activation) and at concentrations of 50.0 µg/plate and higher (with metabolic activation). In tester strains TA 100, TA 1537 and TA 102 toxic effects of the test substance were noted at concentrations of 15.8 µg/plate and higher (without metabolic activation) and at a concentration of 100 µg/plate (with metabolic activation). No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Quaternary ammonium compounds, C12-14-alkyl[(ethylphenyl) methyl]dimethyl, chlorides at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. All criteria of validity were met.

For detailed tables, kindly refer the attached background material section.

Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Quaternary ammonium compounds, C12-14-alkyl[(ethylphenyl) methyl]dimethyl, chlorides did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Quaternary ammonium compounds, C12-14-alkyl[(ethylphenyl)methyl]dimethyl, chlorides is considered to be non-mutagenic in this bacterial reverse mutation assay.

Conclusions:

Under the study conditions, the test substance was determined to be non-mutagenic in the Ames test, with and without metabolic activation.

Executive summary:

An in vitro study was conducted to determine the genotoxic potential of test substance, C12-14 ADEBAC (active: 51.13%), using Ames test, according to OECD Guideline 471 and EU Method B13/14, in compliance with GLP. In two independent experiments, Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 were treated with suspensions of the test substance using the Ames plate incorporation at up to eight dose levels (0.0316 to 100 µg/plate), in triplicate, both with and without metabolic activation (liver S9 in standard co-factors). The doses for experiement 1 and experiement 2 were selected based on preliminary study results. The dose range for experiment 1 and experiment 2 were ranged from 0.0316 to 100 µg/plate (0.0316, 0.100, 0.316, 1.0, 3.16, 10.0, 31.6 and 100 µg/plate) and from 0.050 to 100 µg/plate (0.050, 0.158, 0.50, 1.58, 5.00, 15.8, 50.0 and 100 µg/plate), respectively. No precipitation of the test substance was observed in any tester strain used in experiment I and II (with and without metabolic activation). In experiment I toxic effects of the test substance were observed at concentrations of 10.0 µg/plate and higher (with and without metabolic activation), depending on the particular tester strain. In experiment II toxic effects of the test substance were noted at concentrations of 15.8 µg/plate and higher (without metabolic activation) and at concentrations of 50.0 µg/plate and higher (with metabolic activation), depending on the particular tester strain. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with test substance at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. All criteria of validity were met. Under the study conditions, the test substance was determined to be non-mutagenic in the Ames test, with and without metabolic activation (Schreib, 2017).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Study period:

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

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

Conclusions:

Based on the results of the read across study, the test substance, C12 -14 ADEBAC, can be considered to be non-clastogenic in mammlian cells with and without metabolic activation.

Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the read across substance, C12 -16 ADBAC (active ingredient: >93%), according to OECD Guideline 473 and EU Method B.10 (chromosome aberration test), 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. Under the conditions 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, the test substance, C12 -14 ADEBAC, can be considered to be non-clastogenic in mammlian cells with and without metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Study period:

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

Reason / purpose for cross-reference:

read-across source

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

Conclusions:

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.

Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the read across substance, C12-16 ADBAC (active ingredient: 81.09%), 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 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). Under study conditions, C12 -16 ADBAC was 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 substance study, the test substance, C12 -14 ADEBAC, is not considered to induce mutation in the mammalian cells with and without metabolic activation.

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 genotoxic potential of test substance, C12-14 ADEBAC (active: 51.13%), using Ames test, according to OECD Guideline 471 and EU Method B13/14, in compliance with GLP. In two independent experiments, Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 were treated with suspensions of the test substance using the Ames plate incorporation at up to eight dose levels (0.0316 to 100 µg/plate), in triplicate, both with and without metabolic activation (liver S9 in standard co-factors). The doses for experiement 1 and experiement 2 were selected based on preliminary study results. The dose range for experiment 1 and experiment 2 were ranged from 0.0316 to 100 µg/plate (0.0316, 0.100, 0.316, 1.0, 3.16, 10.0, 31.6 and 100 µg/plate) and from 0.050 to 100 µg/plate (0.050, 0.158, 0.50, 1.58, 5.00, 15.8, 50.0 and 100 µg/plate), respectively. No precipitation of the test substance was observed in any tester strain used in experiment I and II (with and without metabolic activation). In experiment I toxic effects of the test substance were observed at concentrations of 10.0 µg/plate and higher (with and without metabolic activation), depending on the particular tester strain. In experiment II toxic effects of the test substance were noted at concentrations of 15.8 µg/plate and higher (without metabolic activation) and at concentrations of 50.0 µg/plate and higher (with metabolic activation), depending on the particular tester strain. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with test substance at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. All criteria of validity were met. Under the study conditions, the test substance was determined to be non-mutagenic in the Ames test, with and without metabolic activation (Schreib, 2017).

Study 2: An in vitro study was conducted to determine the genotoxic potential of the read across substance, C12-16 ADBAC (active: >93%), according to OECD Guideline 473 and EU Method B.10 (chromosome aberration test), 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 lymphocytesin vitrounder 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. Under the conditions of the study, the read across 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, similar absence of clastogenic potential can be expected for the test substance, C12-14 ADEBAC.

Study 3:An in vitro study was conducted to determine the genotoxic potential of the read across substance, C12-16 ADBAC (active: 81.09%), 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). Under study conditions, C12 -16 ADBAC was 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, similar absence of mutagenic potential can be expected for the test substance, C12-14 ADEBAC.

Justification for classification or non-classification

Based on the absence of genotoxicity in read across studies as well as an Ames test with the test substance, it can be concluded that C12-14 ADEBAC does not warrant classification for genotoxicity according to EU CLP criteria (Regulation 1272/2008/EC).