Reaction products of 2-amino-4,6-dinitrophenol, 4-amino-5-hydroxy 2,7-naphthalenedisulfonic acid, 4-nitrobenzenamine and resorcinol, sodium salts

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 the 8th to the 29th of July, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Justification for Read Across is detailed in the endpoint summary and it is further detailed in the report attached to the IUCLID section 13.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

The test has been conducted following the Environmental Protection Agency, Code of Federal Regulations, Title 40, Subpart F-Genetic Toxicity, Revision July 1st, 1986 "The salmonella Typhimurium Reverse Mutation Assay".

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar substance 01 of Acid Brown 191

IUPAC Name:

Similar substance 01 of Acid Brown 191

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

10.0 µg/plate; 100.0 µg/plate; 333.3 µg/plate; 1000.0 µg/plate; 5000.0 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water bidest- Justification for choice of solvent/vehicle: the solvent was chosen to its solubility properties and its relative nontoxicity for the bacteria.

Controls

Untreated negative controls:

yes

Remarks:

concurrent untreated

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: NaN3 (TA 1535, TA 100); 4-NOPD (TA 1537, TA 1538, TA 98); With metabolic activation: DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
Preincubation period: 6 hours at 37 °C in a shaking water bath
Exposure duration: After solidification the plates wew incubated upside down for 72 hours at 37 °C in the dark

NUMBER OF REPLICATIONS: Two indipendent study, three replicates for each concentration

THE TEST SYSTEM
Charachterization of the salmonella Typhimurium Strains
The strains are derived from S. Thyphimurium 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 well more easily. A further mutation causes a reduction in the activity of an excision repair system. The lattr 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:
TA 1537: his C 3076; rfa-; uvrB-; Frame shift mutations
TA 1538: his D 3052; rfa-; uvrB-; Frame shift mutations
TA 98: his D 3052; rfa-; uvrB-; r-FACTOR; Frame shift mutations
TA 1535: his G 46; rfa-; uvrB-; r-FACTOR; Base-pair substitions
TA 100: his G 46; rfa-; uvrB-; Base-pair substitions
Regular cheking of the propreties of the strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed according to Ames et al.. In this way it is ensured that the experimental conditions set down by ames were fulfilled.
Storage
The strain coltures were stored as stock cultures in ampoules with nutrient broth + 5 % DMS in liquid nitrogen.
Precultures
From the thaved ampoule of the strains 0.5 ml of bacterial suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. This nutrient medium contains per litre:
8 g Difco Nutrient broth
5 g NaCl
The bacterial colture was incubated in a shaking water bath for 6 hours at 37 °C.
Selective Agar
1.5 % vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 ml of this nutrient medium. Sterilizations were performed at 121 °C in an autoclave.
Overlay Agar
The overlay agar contains per litre:
6.0 g Difco Bacto Agar
6.0 g NaCl
10.5 mg L-histidine x Hcl x H2O
12.2 mg biotin
Sterilizations were performed at 121 °C in an autoclave
 
MAMMALIAN MICROSOMAL FRACTION S9 MIX
S9 (preparation)
The S9 liver microsomal fraction was obtained from the liver of 8 – 12 weeks old male Wistar rats, strain WU (SAVO – Ivanovas, med. Versichstierzuchten GmbH) which recived a single i.p. injiection of 500 mg/kg bw Aroclor 1254 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, diluited 1:3 in KCl was centrifuged cold at 9000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 5 ml and stored at – 70 °C. Small numbers of the ampoules are kept at – 20 °C for only several weeks before use. The standardization of the protein content was made using the analysis kit of Bio-Rad Laboratories.
The protein concentration in the S9 preparation is usually between 20 and 45 mg/ml
S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution in a ratio 3:7.
The composition of the cofactor solution was concerntrated to yield the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phospahate
5 mM NADP
in 100 mM sodium-orthp-èhosphate-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.
 
PREEXPERIMENT FOR TOXICITY
To evaluate the toxicity of the test article a prestudy is performed with strains TA 98 and TA 100. 8 concentrations are tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment are the same as described below for the experiment.
Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

DOSE SELECTION
According to the results of this pre-experiment the concentrations applied in the main experiments are chosen. The concentration range covers at least two decadic logarithms.
Appropriate dose levels are selected regarding the characteristics of the test article. At least five different concentrations of the test article are tested. The highest dose level should produce some toxic effects. The maximum concentration is 5000.0 µg/plate, unless limited by toxicity or solubility of the test article.
In case the results of the pre-experiment are in accordance with the criteria described above, these data are reported as a part of the main experiments.

EXPERIMENTAL PERFORMANCE
For each strain and dose level, including the controls, a minimum of three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µl: test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
500 µl S9 mix or S9 mix substitution buffer
100 µl Bacteria suspension
2000 µl Overlay agar

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:
Corresponding background growth on both negative control and test plates
Normal range of spontaneous rerversion tates.
Range of spontaneous reversion frequencies (5,9)TA 1535 (3 - 37); TA 1537 (4 - 31); TA 1538 (12 - 37); TA 98 (15 - 60); TA 100 (75 - 200)
Due to the 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 as positive if either a significant dose
- related increase in the number of revertants nor a significant and reproducible increase for at least one test concentration is induced.A test article producing neither a significant dose
-related 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 positive response is described as follows:
A test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is 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 teest article regardless 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:

other: S. typhimurium TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

other: S. typhimurium TA 1537, TA 1538 and TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: Independent test n. 2

Any other information on results incl. tables

In Strain TA 98 a distinct mutagenic effect occurred without S9 -mix in both experiments. In the presence of metabolic activation no positive response was obtained in Experiment 1. In te experiment 2 a weak increase in the number of revertants was found at 1000 and 5000 µg/plate.

Applicant's summary and conclusion

Conclusions:

Positive with and without metabolic activation.
Under the test condition the substance did induce point mutation by base pair change or frameshifts in the genome of the strains TA 1537, TA 1538, TA 98 and TA 100

Executive summary:

Method

The test has been conducted according to the OECD Guideline 471 and to the EU Method B.14 in order to evaluate the potential of the test item to induce gene mutations according to the plate incorporation test.

During the test the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 have been used. The assay has been performed in two independent experiments, using identical procedures, both with and without microsomal activation. Each concentration, including the controls, has been tested in triplicate. The substance has been tested at the concentrations: 10, 100, 333.3, 1000 and 5000 µg/plate.

Observations

No toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred in any of the test groups with and without metabolic activation.

The plates incubated showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

Up to the highest investigated dose, a significant and reproducible dose-dependent increase in revertant colony numbers was obtained with the strains TA 1537, TA 1538, TA 98 and TA 100. The presence of liver microsomal activation did not influence the genotoxic effect, However the positive response in strain TA 98 was not reproduced in the independent experiment in the presence of S9 mix.

Results

Under the test condition the substance did induce point mutation by base pair change or frameshifts in the genome of the strains TA 1537, TA 1538, TA 98 and TA 100. The test article is considered to be mutagenic in this assay.