Miristalkonium chloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 28, 2001 to March 26, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

Quaternary ammonium compounds, benzyl C12-C16 (even numbered) -alkyldimethyl chlorides

Molecular formula:

C12-14H25-29-(CH3)2-C6H5-N.CL

IUPAC Name:

Quaternary ammonium compounds, benzyl C12-C16 (even numbered) -alkyldimethyl chlorides

Specific details on test material used for the study:

- Physical state: Clear liquid
- Analytical purity: >93%
- Impurities (identity and concentrations): 0.8% Free Amine + Amine hydrochloride and ≤ 0.1% AAS
- Lot/batch No.: DEGE001033
- Expiration date of the lot/batch: January 2002
- Stability under test conditions: The test substance is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five years under standard laboratory conditions.
- Storage condition of test material: Room temperature in the dark
- pH (1% water): 6.5

Method

Target gene:

Histidine

Species / strain

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:

Phenobarbitone/β-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity study: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (With and without metabolic activation)
Main Experiment: Experiment 1 & 2: 0.15, 0.5, 1.5, 5, 15 and 50 µg/plate (With and without metabolic activation)

Vehicle / solvent:

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

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without metabolic activation

Controls 2

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation

Controls 3

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation

Controls 4

Positive controls:

yes

Positive control substance:

other: Nitroquinoline-1-oxide (4NQO)

Remarks:

Without metabolic activation

Controls 5

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

With metabolic activation

Controls 6

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With metabolic activation

Controls 7

Positive controls:

yes

Positive control substance:

other: 1,8-Dihydroxyanthraquinone (DANTHRON)

Remarks:

With metabolic activation

Details on test system and experimental conditions:

Method of application: In agar (direct plate incorporation)
Number of replications: Triplicates

Evaluation criteria:

The test substance was considered positive in the test system if the following criteria were met:
The test substance should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.

Results and discussion

Test results

Key result

Species / strain:

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- The test substance caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains both with and without S9-mix beginning at 15µg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between experiment number, strain type and exposures with or without S9-mix. The test substance was, therefore, tested up to the toxic limit. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation.
- All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Any other information on results incl. tables

Table 1. Cytotoxicity (Number of revertant colonies)

		Test substance concentration (µg/plate)
With/ Without S9-mix	Strain	0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
Without	TA100	91	91	81	83	64	17S	0T	0T	0T	0T	0T
With	TA100	97	105	103	103	83	50S	0T	0T	0T	0T	0T

S=sparse bacterial background lawn

T= toxic, no bacterial lawn

Table 2. Genotoxicity (Mean number of revertant colonies)

Strain	TA100		TA1535		TA102		TA98		TA1537
Test substance concentration (mg/plate)
With S9
+ve control type (concentration (mg/plate))	2AA (1)		2AA (2)		DAN (10)		BP (5)		2AA (2)
Test number	1	2	1	2	1	2	1	2	1	2
+ve control	1772	2317	287	135	886	723	229	251	582	336
-ve control	143	137	17	17	349	308	36	25	12	22
0.15	129	137	13	13	357	315	26	24	13	18
0.5	134	11	10	14	346	313	33	25	15	14
1.5	129	127	15	14	373	307	32	26	16	14
5	142	143	10	13	369	341	32	23	17	16
15	132	0	12	2	363	151	37	11	17	6
50	28	0	10	0	276	0	22	0	10	0
Without S9
+ve control type (concentration (mg/plate))	ENNG (3)		ENNG (5)		MMC (0.5)		4NQO (0.2)		9AA (80)
Test number	1	2	1	2	1	2	1	2	1		2
+ve control	621	464	603	430	854	961	142	126	656		716
-ve control	153	134	12	18	316	336	38	22	16		17
0.15	150	123	13	15	340	308	29	18	18		19
0.5	132	114	11	21	331	339	30	18	17		18
1.5	154	111	19	16	339	232	25	14	15		11
5	145	111	9	10	326	325	28	19	12		18
15	84	0	11	0	320	0	23	0	0		0
50	0	0	0	0	0	0	0	0	0		0

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was found to be non-mutagenic in Ames test 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, according to OECD Guideline 471, EU Method B13/14 and US EPA OPPTS 850.5100 (Ames test), in compliance with GLP. The mutagenic potential was investigated in Salmonella typhimurium strains A1535, TA1537, TA102, TA98 and TA100 with and without metabolic activation. Six dose levels of the test substance for each bacterial strain were tested in triplicate with and without a metabolic activation system. The dose range was determined in a preliminary toxicity assay and was 0.15 to 50 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range, fresh cultures of the bacterial strains and fresh test substance formulations. Additional dose levels were included in both experiments to allow for test substance-induced toxicity and to ensure there were a minimum of four non-toxic doses plated out. The vehicle (sterile distilled water) control plates produced counts of revertant colonies within the normal range. All the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without the S9 -mix. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test substance, either with or without metabolic activation. Under the study conditions, the test substance was found to be non-mutagenic in Ames test with and without metabolic activation (Thompson, 2001).