reaction product of: C.I. Leuco Sulfur Black 1 with (3-chloro-2-hydroxypropyl)trimethylammonium chloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 24. Aug. to 22. Nov. 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Only four strains were used in accordance with the replaced OECD guideline (1983)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

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:

-

EC Number:

424-510-1

EC Name:

-

Cas Number:

220150-59-4

Molecular formula:

not applicable for UVCB substance

IUPAC Name:

Reaction products of Phenol, 2,4-dinitro-, sulfurized, leuco derivatives and (3-chloro-2-hydroxypropyl)trimethylammonium chloride

Method

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

BACTERIAL CELLS

- Source of cells: bacterial strains TA 1535, TA 98, and TA 100 were obtained from Dr. B.N. Ames (University of California, 94720 Berkeley, USA); bacterial strain TA 1537 was obtained from BASF (67063 Ludwigshafen, DE)

- Suitability of cells: the histidine-dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98 and TA 100, the R-factor plasmid pKM 101 carries the ampicillin resistance marker. When summarised, the mutations of the bacterial strains used in this study can be described as follows:
TAI 537: his C 3076; rfa-; uvrB-: frame shift mutations
TA 98: his D 3052; rfa-; uvrB-; R-factor: frame shift mutations
TA1535: his G 46; rfa-; uvrB-: base-pair substitutions
TA 100: his G 46; rfa-; uvrB-; R-factor: base-pair substitutions

- Storage: as stock cultures in ampoules with nutrient broth and 5 % DMSO (MERCK, 64293 Darmstadt, DE) in liquid nitrogen

- Precultures: 0.5 ml bacterial suspension was thawed and transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium (8 g/l Marck Nutient Broth; 5 g/l NaCl; MERCK, 64293 Darmstadt, DE), then incubated in a shaking water bath for 8 hours at 37 °C

MEDIA USED
- Selective agar: 20 ml of 2.0 % Vogel-Bonner-Glucose-Minimal-Agar
- Overlay agar: Merck Agar Agar (6 g/l) (MERCK, 64293 Darmstadt, DE), NaCl (6 g/l), L-histidine x HCl x H20 (10.5 mg/l) and biotin (12.2 mg/l)
- Sterilisation: 121 °C in an autoclave

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

Pre-test: 3.3, 10, 33.3, 100, 333.3, 1000, 2500 and 5000 μg/plate
According to the dose selection criteria, the test article was tested at the following concentrations: 33.3, 100, 333.3, 1000, 2500 and 5000 μg/plate

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

congo red

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

PRE-TEST FOR TOXICITY:
In order to select suitable concentrations of test item for the experimental study and to evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100 in triplicate. The count of revertants per plate at 3.3, 10, 33.3, 100, 333.3, 1000, 2500 and 5000 μg/plate of test
item was compared to count of revertants of a negative control, solvent control and four positive controls (sodium azide (TA 100, without S9), 4-nitro-o-phenylene-diamine (TA 98, without S9) and 2-aminoanthracene (TA 98 and TA 100, with S9).Toxicity of the test article was considered a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or a lower degree of survival of treated cultures. The pre-test did not demonstrate toxicity up to 5000 µg/plate test substance, with or without metabolic activation, in TA 98 or TA 100. The plates with the test item showed normal background growth up to the highest dose, 5000 μg/plate. Therefore, according to the dose selection criteria, the experimental concentrations selected included two logarithmic decdes and were 33.3, 100, 333.3, 1000, 2500 and 5000 μg/plate.

PREPARATIONS:
- Precultures: ampoules of the four strains were thawed and 0.5 ml bacterial suspensions were transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium (8 g/l Merck Nutrient Broth and 5 g/l NaCl; MERCK, 64293 Darmstadt, DE). The bacterial culture was incubated in a shaking water bath for 8 hours at 37 °C.
- Selective agar plates: plates with the minimal agar were obtained from E. Merck, 64293 Darmstadt, DE (lot no. 67087)
- Overlay agar: 6 g/l Merck Agar Agar (MERCK, 64293 Darmstadt, DE), 6 g/l NaCl, 10.5 mg/l Lhistidine x HCl x H2O (MERCK, 64293 Darmstadt, DE), 12.2 mg/l biotin
- Rat Liver S9: S9 liver microsomal fraction was obtained from the livers of 8 to 12 week old male Wistar rats, strain HanIbn (BRL, 4414 Füllinsdorf, CH; weight approximately 220 to 320 g) which received a single injection of 500 mg/kg bw Aroclor 1254 (Antechnika, 76275 Ettlingen, DE) in olive oil 5 days prior. After cervical dislocation, the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate was diluted to 1:3 in KCl and centrifuged cold at 9000 g for 10 minutes at 4 °C.
- Hamster Liver S9: S9 liver microsomal fraction was obtained from the liver of 7 to 8 week old male Syrian golden hamsters (BRL, 4414 Füllinsdorf, CH). After cervical dislocation, the livers of the animals were removed, washed in 0.1 M sodium phosphate buffer pH 7.4, 0.25 M sucrose and 1 mM disodium EDTA in bidistilled water and homogenised. The homogenate was diluted to 1:3 in KCl and centrifuged cold at 9000 g for 10 minutes at 4 °C. A stock of the supernatants containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -80 °C. Small numbers of the ampoules were kept at -20 °C for only several weeks before use. The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories (80939 München, DE): Bio-Rad Protein assay, Catalogue 500 000 6. The protein concentration in the S9 preparations was 30.6 mg/ml in the pre-experiment and in experiment 1; and 30.4 mg/ml in experiment 2.
- Rat S9 mix: S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % (v/v) to yield the following concentrations: 8 mM MgCl2; 33 mM KCl; 5 mM glucose-6-phosphate; 5 mM NADP; in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
- Hamster S9 mix: S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % (v/v) to yield the following concentrations: 8 mM MgCl2; 33 mM KCl; 20 mM glucose 6-phosphate; 2.8 units/ml glucose-6-phosphate dehydrogenase; 4 mM NADP; 2 mM NADH; 2 mM FMN; in 100 mM sodium-orthophosphate-buffer, pH 7.4.

METHOD:
i) Each test strain was tested both with and without metabolic activation, at all 5 test item concentrations, with an appropriate positive control, and with both a negative control and a solvent, in triplicate. The following materials were mixed in a test tube and incubated at 37 °C for 60 minutes:
- 100 μl test item at each dose level (33.3, 100, 333.3, 1000, 2500 and 5000 μg/plate), solvent (negative control), or reference mutagen solution (positive control);
- 500 μl S9 mix ("with metabolic activtion") or S9 mix substitution buffer ("without metabolic activation")
- 100 μl bacterial suspension (TA 1535, TA 1537, TA 98 or TA 100)
ii) Following test tube incubation, 2000 µl overlay agar was added to each test tube then plated.
iii) Plates were allowed to solidify, then were incubated upside down for at least 48 hours at 37 °C inthe dark.
iv) Colonies were counted using the AUTOCOUNT counter (Artek Systems Corporation, BIOSYS GmbH, 61184 Karben, DE). Obscured plates (due to colour of test item) were counted manually.

Rationale for test conditions:

The most widely used assays for detecting gene mutations are those using bacteria. They are relatively simple and rapid to perform, and give reliable data on the ability of an agent to interact with DNA and produce mutations. Especially for axo dyes, the exogenous metabolic system described by Prival and Mitchell is preferred. In spite of great differences between bacterial and eukaryotic cells with respect to structure and function there is a relationship between mutagenicity in bacteria and carcinogenicity in mammals.
Reverse mutation assays determine the frequency with which an agent repairs or suppresses the effect of the forward mutation. The genetic target presented to an agent is therefore small, specific and selective. Several bacterial strains, or a single strain with multiple markers are necessary to overcome the effects of mutagen specificity. THe reversion of bacteria from growth-dependence or a particular amino acid to growth in the absence of that amino acid (reversion from auxotrophy to prototrophy) is the most widely used marker.
The Salmonella typhimurium histidine (his) reversion system measures his- to his+ reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 1535 and TA 100) and frameshift (TA 1537 and TA 98) mutatations.

Evaluation criteria:

Range of Spontaneous Reversion Frequencies:
TA 1535: 10 to 29
TA 1537: 5 to 28
TA 98: 15 to 57
TA 100: 77 to 189

A test item is considered positive if either a dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced. A test item producing neigher a dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points in considered non-mutagenic in this system.
A test item is considered mutagenic if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 at least three times higher as compared to the spontaneous reversion rate. Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless of whether the highest dose induced the above described enhancement factors or not.

Statistics:

No appropriate statistical method is available.

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:

no cytotoxicity

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:

no cytotoxicity

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- No toxic effects, evidenced by a reduction in the number of revertants, occurred in the test groups with and without matabolic activation at the highest investigated dose.
- The plates incubated with the test article showed normal background growth up to 5000 µg/plate, with and without S9 mix, in all strains used.
- No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological ralavance.
- The strain TA 98 showed an enhancement factor of 2.0 at 2500 µg/plate in the presence of metabolic activation in the first experiment. This increase was judged as irrelevant since it only occurred at a single concentration in the first experiment and could not be reproduced at any concentration in the second experiment.
- Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

Any other information on results incl. tables

PRELIMINARY EXPERIMENT FOR TOXICITY

To evaluate the toxicity of the test item, a pre-study was performed with strains TA 98 and TA 100. The plates with the test item showed normal background growth up to 5000 µg/plate in strain TA 98 and TA 100. According to the dose selection criteria, the test item was subsequently tested in the main study at the following concentrations: 33.3, 100, 333.3, 1000, 2500 and 5000 µg/plate.

Applicant's summary and conclusion

Conclusions:

The test item did not induce gene mutations by base pair changes or frameshifts in genome of strains used of Salmonella typhimurium in the absence or presence of metabolic activation, under the reported experimental conditions.

Executive summary:

The potential of the test item to induce point mutations by base pair changes or frameshifts was evaluated in an experimental study according to the OECD Guideline 471 (1983) and the EU Method B.14 (1992).

8 concentrations of the test item (3.3, 10, 33.3, 100, 333.3, 1000, 2500 and 5000 µg/plate) were tested for toxicity in strains TA 98 and TA 100 in triplicate using the pre-incubation method in a pre-study. Based on the results obtained, concentrations of 33.3, 100, 333.3, 1000, 2500 and 5000 µg/plate were selected for the main study.

Two independent main experiments, utilsing the pre-incubation and plate-incoporation methods, were performed to Salmonella typhimurium strains TA 1535, TA 1537, TA 100 and TA 98, both with and without metabolic activation (S9 Mix). In each experiment, 3 agar plates were prepared for each strain and dose level, including controls.

In the pre-study the plates incubated with the test item showed normal background growth up to 5000 μg/plate, both with and without S9 mix, in all strains used. No toxic effects occurred at any of the dosages, any strain, with or without metabolic activation, in either the first or second experiment. No substantial increase in revertant colony numbers was observed in any S. typhimurium strain; with or without metabolic activation. The strain TA 98 showed an enhancement factor of 2.0 at 2500 µg/plate in the presence of metabolic activation in the first experiment, however this increase was judged as irrelevant since it only occurred at a single concentration in the first experiment and could not be reproduced at any concentration in the second experiment, and was not dose-related. Appropriate reference mutagens used as positive controls showed a distinct increase in induced revertant colonies.

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce point mutations by base pair changes and frameshifts in the genome of the strains used. Therefore, the substance is not considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.