Quaternary ammonium compounds, bis(hydrogenated tallow alkyl)dimethyl, acetates

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Comparable to guideline study with acceptable restrictions: the experiment was not repeated in a 2nd independent assay to confirm the lack of mutagenic effect. Cover page of the report is missing.

Justification for type of information:

The primary component of all substances provide complete coverage of 68334-33-8. 68334-33-8 shares high structural similarity with 61789-80-8, 68783-78-8, 107-64-2, and 112-02-7. As 68334-33-8 is a UVCB its components encapsulate the other substances except for the counter ion (Cl-). In solution, the counter ions will dissociate from the parent structures. Therefore, we are comparing substances of equivalent nature. CAS 107-64-2 represents the C18 boundary of the 61789-80-8. Ignoring the salt component CAS 61789-80-8 is equivalent to CAS 68334-33-8. 68783-78-8 is a worst case of both 68334-33-8 and 61789-80-8 since it is unsaturated and the SP2 carbon-carbon bonds are of higher reactivity and a more likely site of metabolic activation. The primary component of CAS 112-02-7 is a substructure of all the other substances. Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

The primary component of all substances provide complete coverage of 68334-33-8. 68334-33-8 shares high structural similarity with 61789-80-8, 68783-78-8, 107-64-2, and 112-02-7. As 68334-33-8 is a UVCB its components encapsulate the other substances except for the counter ion (Cl-). In solution, the counter ions will dissociate from the parent structures. Therefore, we are comparing substances of equivalent nature. CAS 107-64-2 represents the C18 boundary of the 61789-80-8. Ignoring the salt component CAS 61789-80-8 is equivalent to CAS 68334-33-8. 68783-78-8 is a worst case of both 68334-33-8 and 61789-80-8 since it is unsaturated and the SP2 carbon-carbon bonds are of higher reactivity and a more likely site of metabolic activation. The primary component of CAS 112-02-7 is a substructure of all the other substances. Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.

Reason / purpose for cross-reference:

read-across source

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:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 25000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

experiment not repeated in a 2nd independent assay to confirm the lack of mutagenic effect.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

295-835-2

EC Name:

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

Cas Number:

92129-33-4

Molecular formula:

R2N+(CH3)2, Cl- with R is fatty alkyl with chainlengths C16-C18 (even numbered)

IUPAC Name:

N-C16-C18(even numbered)-alkyl-N,N-dimethyl-C16-C18(even numbered)-alkyl-1-aminium chloride

Details on test material:

- Name of test material (as cited in study report): Arquad 2HT-75 737

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from the livers of Aroclor 1254 induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

Toxicity study: 0, 4, 20, 100, 500, 2500 and 10000 µg/plate
Mutagenicity study Salmonella strains: 0, 4, 20, 100, 500 and 1000 µg/plate
Mutagenicity study WP2uvrA strain: 0, 4, 20, 100, 500 and 2500 µg/plate

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: See table 1

Details on test system and experimental conditions:

Bacteria were grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No. 2/litre) at 37°C. The amount of bacteria in suspension was checked by nephelometry. Stock cultures were stored at -80°C.

Preliminary toxicity tests were performed with all tester strains using a small number of plates, results were used to determine dose levels for the mutagenicity study. A reduced rate of spontaneously occurring colonies as well as visible thinning of the bacterial lawn (assessed microscopically) were used as indicators for toxicity. In combination with the mutagenicity study, toxicity testing was performed as follows: 0.1 ml test solution was mixed with 0.1 ml of a 10exp-6 dilution of the overnight culture of TA100 and plated with histidine and biotin rich top agar in triplicate. The solvent control was compared with the number of colonies per plate.

For the mutagenicity study, top agar was prepared for the Salmonella strains by mixing 100 ml agar (0.6%) agar, 0.5% NaCl) with a 5 ml 1.0 mM histidine and 1.0 mM biotin solution. With E.coli histidine was replaced by tryptophan ((5 ml, 0.5 mM). The following was added (in order) to 2 ml of molton top agar at 45°C: 0.1 ml test compound solution, 0.1 ml overnight nutrient broth culture of bacterial tester strain, 0.5 ml S9 mix or buffer. After mixing the liquid was poured onto a petridish with minimal agar (1.5% agar, Vogel-Bonner E medium with 2% glucose). After incubation for 48 to 72 hours at 37°C in the dark, revertant colonies were counted. All concentrations were plated in triplicate.

Evaluation criteria:

Toxicity was assessed by a thinning of the bacterial lawn. Mutagenicity was assessed by comparing the number of revertant colonies in the treated cultures with those of the controls.

Statistics:

No formal statistical analysis was carried out.

Results and discussion

Test results

Test results 1

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:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 25000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Sterility of the S9 mix and test compound was confirmed, and postive and negative control cultures gave the expected number of colonies.
The test material was very toxic to the bacterial strains at concentrations of 2500 µg/plate and higher. Thinning of the bacterial lawn and/or a reduction in the number of colonies was observed at 1000, 2500 and 10000 µg/plate. For mutagenicity testing, 1000 µg/plate was chosen as the top dose for Salmonella strains, and 2500 µg/ml was chosen as the top dose for WP2uvrA.

The test material did not cause a significant increase in the number of revertant colonies with any of the tester strains both in the presence and absence of S9 mix.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the test substance did not show any mutagenic activity in the five Salmonella typhimurium strains tested with and without metabolic activation.

Executive summary:

The potential of the test substance Dioctadecyldimethylammonium chloride (90% active in isopropanol/water) to induce reverse mutation in bacteria was assessed using five strains of Salmonella typhimurium and Escherichia coli in a test similar to OECD guideline 471. The study predates the principles of Good Laboratory Practice. A preliminary toxicity test was performed to define the dose-levels of the test substance to be used for the mutagenicity study . The test item was then tested in one independent experiment both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. The experiment was performed according to the direct plate incorporation method. After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

The preliminary assay with or without metabolic activation showed that the test substance demonstrated a potent toxicity from 10 000 to 2 500 µg/plate in all Salmonella strains tested. Under these conditions, the dose of 1000 µg/plate was retained as the maximum dose tested for the mutagenicity assay in Salmonella strains. In Escherichia Coli WP2uvrA, the top dose was 2500 µg/plate.

 

In the main experiment, the number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. No precipitate was observed in the petri plates when scoring the revertants at all dose-levels. Without metabolic activation, toxicity was observed at the dose level of 1000 µg /plate for TA 1537, TA 1538, TA 98 and TA 100 strains. In Escherichia Coli WP2uvrA, toxicity was noted at 2500 µg /plate. With metabolic activation, cytotoxicity was limited to the strain TA 1537 which exhibited toxicity at the highest dose-level of 1000 µg/plate. No significant increase in the mean number of revertants was noted in the five Salmonella typhimurium strains and Escherichia Coli tested in the presence of the test substance neither with nor without metabolic activation. It was concluded that Dioctadecyldimethylammonium chloride was not mutagen under the conditions of the study