Reaction products of disodium 3,5-diamino-2-[(2-sulfonatophenyl)diazenyl]benzoate and  diazotised sodium 2-[(4-aminophenyl)sulfonyl]ethyl sulfate

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:

Study initiation date - 04 February 2003; Experiment start date - 05 February 2003; Experiment end date - 21 February 2003; Study completion date - 17 March 2003;

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

2000/32/EC, Annex 4D

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identity: FAT 40’810/A
Batch No.: WP 6/02
Aggregate State: At Room Temperature: Solid (powder)
Colour: Brown
Purity: approximately 75%
Solvent: Dimethylsulfoxide (DMSO)
Stability in Solvent: In view of the short term nature of studies of this type, no analyses of stability or achieved concentration were carried out on the preparations of the test item or reference items either prior to or after addition to the bacterial cultures.
Stability of the Test Item: 7 days in water, saline, polyethylene glycol, or carboxymethylcellulose, 1 day in vaseline or FCA
Storage: Room temperature (20 ±3 °C), protect from light.
Expiry Date: December 12, 2009

Specific details on test material used for the study:

Identity: FAT 40810/A
Batch: WP 6/02
Purity: approx. 75 %
Appearance: Solid, dark brownish powder
Expiration date: 12 December 2010
Storage: At room temperature at about 20 °C

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

The histidine-auxotrophic strains of Salmonella typhimurium TA 98 and TA 1535 were obtained from Prof. B. Ames, Berkeley, USA (1983). Strain TA 100 was obtained from Dr. M. Schupbach, Hoffmann-La Roche Limited, Basel, Switzerland (1986). Strain TA 1537 was obtained from Dr. S. Albertini, Hoffmann-La Roche Limited, Basel, Switzerland (1994). The tryptophan-auxotrophic strain of Escherichia coli (WP2 uvrA) was obtained from the National Collection of Industrial Bacteria, Aberdeen, Scotland (1977). The strain cultures were stored as stock cultures in ampoules with nutrient broth + DMSO (10 ml + 1ml) in a deep freezer at about -80 °C.

Metabolic activation:

with and without

Metabolic activation system:

S9 (Preparation in-house)
Rat-liver post mitochondrial supernatant (S9 fraction) was prepared in advance from male rats (HanBrl:WIST SPF), delivered by RCC Ltd, Animal Breeding and Biotechnology, Füllinsdorf, Switzerland. The animals were treated with Aroclor 1254 (Analabs Inc., delivered from Antechnika, Karslruhe, Germany), 500 mg/kg, i.p. 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCI. The homogenate was centrifuged for 15 minutes at 9000x g and the resulting supernatant (S9 fraction) was stored at approximately -80 °C for no longer than one year. The protein content of the S9 fraction was 31.31 mg/ml.

S9 Mix
On day of experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 10 % v/v in the mixture. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCI
5 mM Glucose-6-phosphate
4 mM NADP
in 100 mM sodium phosphate-buffer, pH 7.4.
Before starting the experiment the S9 mix was sterile filtered and stored in a refrigerator. The S9 mix preparation was performed according to Ames et al.

Test concentrations with justification for top dose:

Concentration range in the main test (with and without metabolic activation): 312.5, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

Solvent: dimethylsulfoxide (DMSO)

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100 and TA1535, without S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 without S9

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 without S9

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

E. coli WP2 uvrA without S9

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

TA1535 with S9

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA98, TA100, TA1537 and E. coli WP2 uvrA with S9

Details on test system and experimental conditions:

Storage: The strain cultures were stored as stock cultures in ampoules with nutrient broth + DMSO (10 ml + 1ml) in a deep freezer at about -80 °C.
Precultures: 60 µl aliquots from a frozen stock were grown in 60 ml nutrient broth medium in a 300 ml Erlenmeyer flask (Nutrient broth No. 2, Oxoid Ltd., Basingstoke, U.K) for 8 hours by orbital shaking at 37 °C, 130 - 140 rounds per minute and then used for the experiment. The bacterial cultures were incubated in a temperature / time controlled incubator.
Selective Agar: The plates with the selective agar (minimal agar plates) were made in-house. Each Petri dish contained about 20.0 ml of minimal agar (1.5 % agar supplemented with 2 % salts of the Vogel-Bonner Medium E and 2 % glucose). The agar used was Select AGAR, GIBCO BRL, Switzerland. Glucose was delivered from Merck, Germany [D(+)glucose, anhydrous]. The Vogel-Bonner Medium E was prepared in-house.
Overlay Agar: The overlay agar contained per litre: 6.0 g GIBCO BRL Select Agar and 6.0 g NaCl (Merck, Germany). Sterilisation was performed at 121 °C in an autoclave, cooled down to 50 °C and dispensed into glass bottles. On day of test performance the agar was molten in a water bath and 10% aliquots (v/v) of 0.5 mM L-histidine / 0.5 mM d-biotin for Salmonella strains or 0.5 mM tryptophan, dissolved in bidistilled water, for E. coli strains were added sterile filtered.
Mammalian Microsomal Fraction S9 Mix: The bacteria used in this assay 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 in-house): Rat-liver post mitochondrial supernatant (S9 fraction) was prepared in advance from male rats (HanBrl:WlST SPF), delivered by RCC Ltd, Animal Breeding and Biotechnology, Füllinsdorf, Switzerland. The animals were treated with Aroclor 1254 (Analabs lnc., delivered from Antechnika, Karslruhe, Germany), 500 mg/kg, i.p. 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCI. The homogenate was centrifuged for 15 minutes at 9000x g and the resulting supernatant (S9 fraction) was stored at approximately -80 °C for no longer than one year. The protein content of the S9 fraction was 31.31 mg/ml.
S9 Mix: On day of experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 10 % v/v in the mixture. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgCI2 ; 33 mM KCI ; 5 mM Glucose-6-phosphate; 4 mM NADP in 100 mM sodium phosphate-buffer, pH 7.4. Before starting the experiment the S9 mix was sterile filtered and stored in a refrigerator. The S9 mix preparation was performed according to Ames et aI.


NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments:
Two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation) and preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period:
48 hours


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Background growth inhibition.


METHODS FOR MEASUREMENTS OF GENOTOXICIY
: A biologically relevant increase in the number of revertants.

Evaluation criteria:

- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA1535 and TA1537) the colony count of the corresponding solvent control is observed.
- A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
- An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

A statistical analysis was not required. At present the use of statistical methods concerning this particular test system is not generally recommended.

Results and discussion

Test results

Species / strain:

other: Salmonella typhimurium strains TA 100, TA 1535, TA 98, TA 1537, and the Escherichia coli strain WP2 uvrA.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

(< 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations: A precipitation of the test item was not observed on the surface of any agar plate.

Applicant's summary and conclusion

Conclusions:

FAT 40810/A did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The test item FAT 40810/A was assessed for its potential to induce gene mutations according to the plate incorporation test (first experiment with and without metabolic activation, second experiment without activation) and the pre-incubation test (second experiment with metabolic activation) using Salmonella typhimurium strains TA 100, TA 1535, TA 98, TA 1537, and the Escherichia coli strain WP2 uvrA. This study was conducted in accordance with OECD test guideline 471 and EU method B.13/14. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, the negative (solvent control) and the positive controls were tested in triplicate. The test item was tested at the following concentrations: 312.5, 625, 1250, 2500 and 5000 µg/plate. In both mutagenicity tests normal background growth was observed with all strains at all concentrations tested. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No precipitation of the test item was observed on the surface of the agar plates. No substantial increase in revertant colony numbers of any of the five tester strains occurred following treatment with FAT 40’810/A at any concentration level, neither in the presence nor in the absence of an metabolic activation system. 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. They showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the given experimental conditions reported, FAT 40810/A did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.