Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Acid Blue 185 is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 25, 1994 to January 30, 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
None
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 4 tester strains tested; no tester strain to detect crosslinking mutagens included
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
EU Method B.14
Deviations:
no
Principles of method if other than guideline:
Guidelines followed
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Name: FAT 20'063/C (Lanasol blau 8G roh trocken)
Batch No.: 3.46
Aggregate State at Room Temperature: solid
Colour: dark blue
Purity: 81.9 % (active ingredient)
Stability: Pure: April, 1999
Storage: room temperature
Expiration Date: April, 1999

On the day of experiment, the test article was dissolved in aqua bidest.. The solvent was chosen because of its solubility properties.
Target gene:
Histidine requiring Salmonella strains
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction
Test concentrations with justification for top dose:
33.3; 100; 333.3; 1000; 2500; and 5000 µg/plate (active ingredient)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Without S9: sodium azide (NaN3) for TA 1535, TA 100; For 4-nitro-o-phenylene-diamine (4-NOPD) for TA 1537, TA 98; With S9: 2-aminoanthracene (2-AA) for all the strains
Details on test system and experimental conditions:
THE TEST SYSTEM
Characterisation of the Salmonella typhimurium Strains
The strains are derived from S. typhimurium strain LT2 and due to a mutation in the histidine locus are histidine dependent. 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. In summary, the mutations of the TA strains used in this study can be described as follows:
Salmonella typhimurium
TA 1537: his C 3076; rfa-; uvrB-; : frame shift mutations
TA 98: his D 3052; rfa-; uvrB-;R-factor: frame shift mutations
TA 1535: his G 46; rfa-; uvrB-; : base-pair substitutions
TA 100: his G 46; rfa-; uvrB-;R-factor: base-pair substitutions

Regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in C C R according to Ames et al.. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

Storage
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.

Precultures
From the thawed ampoules of the strains 0.5 ml suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 ul ampicillin was added to the strains TA 98 and TA 100.

This nutrient medium contains per litre:
8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt)
5 g NaCl (MERCK, D-64293 Darmstadt)
The bacterial culture was incubated in a shaking water bath for 10 hours at 37 °C.

Selective Agar
2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 ml of this nutrient medium.
Sterilisations were performed at 121 °C in an autoclave.

Overlay Agar
The overlay agar contains per litre:
6.0 g Merck Agar Agar*
6.0 g NaCl*
10.5 mg L-histidine x HCl x B^O*
12.2 mg biotin*

* (MERCK, D-64293 Darmstadt)
Sterilisations were performed at 121 °C in an autoclave.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
The bacteria used in these assays do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.

S9 (Preparation by C C R)
The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats strain Wistar Hanlbm (BRL, CH-4414 Füllinsdorf; weight approx. 220 - 320 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, D- 76275 Ettlingen, F.R.G.) in olive oil 5 days previously.

After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate was diluted 1+3 in KCl and centrifuged cold at 9.000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -80 °C. Small numbers of the ampoules are kept at -20 °C for only one week before use. The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-80939 München: Bio-Rad protein assay, Catalogue 500 000 6.

The protein concentration in the S9 preparation was 31.8 mg/ml (lot 270694) .

S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15% v/v. The composition of the co-factor solution was concentrated to yield the following concentrations in the S9 mix:

8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.
Evaluation criteria:
The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative controls and test plates
- normal range of spontaneous reversion rates.

Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial
assays at this time.

A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a-significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered
non-mutagenic in this system.
A significant response is described as follows:
A test article 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 and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.
Statistics:
No appropriate statistical method is available.
Key result
Species / strain:
other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and 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

RESULTS

PRE-EXPERIMENT FOR TOXICITY

The plates with the test article showed normal background growth up to 5000.0 µg/plate in strain TA 98 and TA 100, respectively. 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 (active ingredient).

No significant toxic effects, evidenced by a reduction in the number of revertants, occurred in any of the test groups with and without metabolic activation up to 5000 µg/plate.

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 FAT 20'063/C (Lanasol blau 8G roh trocken) at any dose 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 relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusions:
FAT 20'063/C is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.
Executive summary:

A study was performed to investigate the potential of FAT 20'063/C (Lanasol blau 8G roh trocken) to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The assay was performed according to OECD Guideline 471 and EU Method B.14, in two independent experiments, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33.3; 100.0; 333.3; 1000; 2500; and 5000 µg/plate (active ingredient). No significant toxic effects, evidenced by a reduction in the number of revertants, occurred in any of the test groups with and without metabolic activation up to 5000 µg/plate. 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 FAT 20'06 3/C at any dose 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 relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, FAT 20'063/C (Lanasol blau 8G roh trocken) is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A study was performed to investigate the potential of FAT 20'063/C (Lanasol blau 8G roh trocken) to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The assay was performed according to OECD Guideline 471 and EU Method B.14, in two independent experiments, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33.3; 100; 333.3; 1000; 2500; and 5000 µg/plate (active ingredient). No significant toxic effects, evidenced by a reduction in the number of revertants, occurred in any of the test groups with and without metabolic activation up to 5000 µg/plate. The plates incubated with the test article showed normal background growth up to 5000.0 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 FAT 20'06 3/C (Lanasol blau 8G roh trocken) at any dose 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 relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, FAT 20'063/C (Lanasol blau 8G roh trocken) is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Justification for classification or non-classification

Based on the result of a bacterial reverse mutation assay, the substance does not warrant classification according to the CLP (Regulation 1272/2008) criteria.