Dimethyldioctylammonium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are no studies available for the assessment of in vitro genotoxicity of the substance. However, there are three reliable in vitro studies available for a structurally similar substance. The substance (at concentrations up to 50 µg/plate) was found to be negative for mutagenicity in the reverse mutation assay (Ames) employing Salmonella typhimurium strains TA1535, TA1537, TA98, TA102 and TA100 in the presence and absence of a metabolic activation system. The substance was found to be negative for clastogenicity in a chromosome aberration test using Chinese hamster ovary cells in the presence and absence of a metabolic activation system. The substance was found to be negative in the forward mutation assay in CHO cells at the HGPRT locus in the presence and absence of a metabolic activation system.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 August 1986 - 18 Spetember 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Read-across

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1B4

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 from Aroclor 1254 induced rats.

Test concentrations with justification for top dose:

The concentration of the test substance employed was based on the results from a toxicity test. The levels selected were 2, 4, 8 and 16 µg/ml (with S9 mix) and 1, 2, 4 and 8 µg/ml (without S9 mix).

Vehicle / solvent:

Distilled water.

Untreated negative controls:

yes

Remarks:

Vehicle control - distilled water

Negative solvent / vehicle controls:

no

True negative controls:

yes

Remarks:

Untreated control cultuure

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Mitotic inhibitor was colcemid (N-deacetyl-N-methylcolchicine)

Evaluation criteria:

All structural chromosome aberrations were assigned artifical lesion or break scores and the aberrations yield quantified as follows:
i) Chromatid and chromosome (isochromatid) gaps, breaks and acentric fragments were counted as one lesion each, indicating that a single event had generated the damage.
ii) Chromatid and chromosome complexes, such as exchanges, rings and dicentrics were designated 2 lesions each, indicating that 2 separate breaks has occurred. A cell containing a rearrangement and an associated fragment was, however, scored as having 2 and not 3 lesions, due to the likely shared origin of this damage.
iii) Metaphase with chromosomes showing multiple and extensive aberrations were awarded an arbitary value of 10 lesions per cell.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The substance was found to be negative for clastogencity in a chromosome aberration test using Chinese hamster ovary cells in the presence and absence of a metabolic activation system.

Executive summary:

The substance was tested for clastogenic potential using Chinese hamster ovary cells in vitro in the presence and absence of a metabolic activation system (liver S9 from Aroclor 1254 induced rats). There was no evidence of chromosomal aberrations and no indication of chromosomal ploidy changes following exposure to the substance at concentrations of 2, 4, 8 and 16 µg/ml (with S9 mix) and 1, 2, 4 and 8 µg/ml (without S9 mix). It was concluded that the substance was negative for clastogencity in a chromosome aberration test using Chinese hamster ovary cells in the presence and absence of a metabolic activation system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 March 2001 - 22 March 2001

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:

not specified

GLP compliance:

yes

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:

10% liver S9 in standard co-factors.

Test concentrations with justification for top dose:

Doses were selected based on a range finder study.
using 0.05 to 15 µg/plate without S9.
Dose range finder using 0.15 to 50 µg/plate with S9.

Vehicle / solvent:

Dimethyl sulphoxide.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 1,8-Dihydroxyanthraquinone

Evaluation criteria:

The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The substance (at concentrations up to 50 µg/plate) was found to be negative for mutagenicity in the reverse mutation assay employing Salmonella typhimurium strains TA1535, TA1537, TA98, TA102 and TA100 in the presence and absence of a metabolic activation system.

Executive summary:

In the in vitro genotoxicity study (Ames test) the substance was tested for mutagenicity in Salmonella typhimurium strains TA1535, TA1537, TA98, TA102 and TA100. Concentrations of up to 50 μg/plate were tested. No evidence of mutagenic activity was seen at any concentration of the substance in the presence and absence of a metabolic activation system. It was concluded that the substance showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 December 1987 - 18 August 1988

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)

Version / remarks:

1984

Deviations:

yes

Remarks:

The recommended performance of an independent repeat experiment for the confirmation of results was not included. The author stated that this deviation did not affect the quality/integrity of the study or the intepretation of the results in this report.

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

Version / remarks:

1986

Deviations:

yes

Remarks:

The recommended performance of an independent repeat experiment for the confirmation of results was not included. The author stated that this deviation did not affect the quality/integrity of the study or the intepretation of the results in this report.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

Substance was stored at room temperature in the dark.
Lot number: B-1889.
Solubility was obtained in sterile deionised water at 50 mg/ml.
Analysis of selected primary stocks were performed within 24 hours of preparation.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Hypodiploid CHO-K1-BH4 cell line

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 from animals treated with Aroclor 1254.

Test concentrations with justification for top dose:

The concentrations employed in the mutation assay was based on the findings from the rangefinder assay.
Absence of S9: 1 µg/mL to 13 µg/mL
Presence of S9: 1 µg/mL to 40 µg/mL

Vehicle / solvent:

Deionised water (sterile).

Untreated negative controls:

yes

Remarks:

Media alone. For the assays in the presence of S9 the media was mixed with the S9 mix.

Negative solvent / vehicle controls:

yes

Remarks:

Cells were exposed to culture media containing water and no substance.

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

other: 5-Bromo-2-deoxyuridine

Evaluation criteria:

The 95% confidence interval must be met as one criterion for considering the substance to be active at a particular dose level. The mutant frequency must meet or exceed 15 x 10-6 in order to compensate for random fluctuations in the 0 to 10 x 10-6 background mutant frequencies that are typical for this assay.
A dose-related or toxicity-related increase in mutant frequency should be observed.
If an increase in mutant frequency is observed for a single dose near the highest testable toxicity and the number of mutant colonies is more than twice the value needed to indicate a significant response, the substance generally will be considered to be mutagenic. Smaller increases at a single dose near the highest testable toxicity will require confirmation by a repeat assay.
Applied concentration or toxicity (percent survival) can be used to establish whether the mutagenic activity is related to an increase in effective treatment.
A substance is evaluated as nonmutagenic in a single assay only if the minimum incrase in mutant frequency is not observable for a range of applied concentrations that extends to concentrations causing about 10% to 15% survival or extends to a concentrations will normally be 5 mg/ml for water soluble materials. If a repeat assay does not cofirm aan earlier, minimal response the substance is evaluated as nonmutagenic in this assay system.

Statistics:

The statistical tables provided by Kastenbaum and Bowman (1970) are used to determine whether the results at each dose level are significantly different from the vehicle controls at 95% or 99% confidence intervals. This test compares variables distributed according to Poissonian expectations by summing up the probabilities in the tails of two binomial distributions.The 95% confidence interval must be met as one criterion for considering the substance to be actived at a particular dose level.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The substance was found to be negative in the forward mutation assay in CHO cells at the HGPRT locus in the presence and absence of a metabolic activation system.

Executive summary:

The substance was evaluated for its potential to induce forward mutations at the HGPRT locus in the CHO-K1 -BH4 CHO cell line as assessed by colony growth in the presence of 6 -thioguanidine (TG), in the presence and absence of a metabolic activation system (S9). The substance was found to be negative in the assay in the presence and absence of a metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

There are no studies available for the assessment of in vivo genotoxicity of the substance. However, there is a reliable in vivo study available for a structurally similar substance. The substance was found to be negative for chromosomal damage in rat bone marrow in the in vivo cytogenetic test when administered orally by gavage.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 September 1986 - 20 January 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Read-across

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

1984

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1984

GLP compliance:

yes

Type of assay:

other: Micronucleus assay

Specific details on test material used for the study:

Batch number: E06130085
Purity: 50.3%

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CRL UK
- Assigned to test groups randomly: yes
- Fasting period before study: overnight prior to dosing
- Housing: plastic disposable cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Air changes (per hr): 30
- Photoperiod (hrs dark / hrs light): 12 hours artifical light/day

Route of administration:

oral: gavage

Vehicle:

Sterile distilled water.

Details on exposure:

All animals in all groups were dosed by oral gavage with a standard volume of 20 ml/kg bw except that those receiving cyclophosphamide were dosed by IP injection.

Duration of treatment / exposure:

Single administration of dose.

Frequency of treatment:

Once.

Post exposure period:

48 hours

Dose / conc.:

600 mg/kg bw/day (nominal)

No. of animals per sex per dose:

35/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
40 mg/kg bw/day
Prepared in a solution in sterile 0.9% saline at a concentration of 2.0 mg/ml.

Tissues and cell types examined:

Both femurs were dissected from the animals and the proximal epiphysis was remoaved and the marrow eluted.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A preliminary toxicity test was conducted to evaluate the toxicity of the substance at various doses. The dose selcted for the main test was based on the lowest dose that did not result in any toxicity.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Sampling time (hrs)	Treatment	Doseage (mg/kg bw)	Incidence of aberrant cells (%)
			Excluding gap damage	Including gap damage
6	Vehicle P0151	- 600	0 0	0 0
24	Vehicle P0151 Cyclophosphamide	- 600 40	0 0 5.6	0 0 5.6
48	Vehicle P0151	- 600	0 0	0 0

Conclusions:

The substance was found to be negative for chromosomal damage in rat bone marrow in the in vivo cytogenetic test when administered orally by gavage.

Executive summary:

The substance was assessed for its potential to induce chromosomal damage in rats dosed by oral gavage at a dose of 600 mg/kg bw. The animals were observed for 48 hours for any signs of toxicity. Two hours prior to sacrifice the animals each received a dose of colchicine to arrest cells in the metaphase stage of cell division. At the end of the observation period the animals were terminated by cervical dislocation and the bone marrow from the femurs were removed for analysis. The substance was found to be negative for chromosomal damage in rat bone marrow in the in vivo cytogenetic test when administered orally by gavage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the findings of three reliable in vitro and one reliable in vivo genotoxicity studies conducted on a structurally similar substance, classification of the substance is not justified.