1,2-dihydro-6-hydroxy-1,4-dimethyl-2-oxo-5-[[3-[(phenylsulphonyl)oxy]phenyl]azo]nicotinonitrile

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:

14 January 1999 to 25 February 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 4 tester strains tested; no tester strain to detect cross-linking mutagens was included

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

only 4 tester strains tested; no tester strain to detect cross-linking mutagens was included

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,2-dihydro-6-hydroxy-1,4-dimethyl-2-oxo-5-[[3-[(phenylsulphonyl)oxy]phenyl]azo]nicotinonitrile

EC Number:

261-693-5

EC Name:

1,2-dihydro-6-hydroxy-1,4-dimethyl-2-oxo-5-[[3-[(phenylsulphonyl)oxy]phenyl]azo]nicotinonitrile

Cas Number:

59312-61-7

Molecular formula:

C20H16N4O5S

IUPAC Name:

3-[(5-cyano-2-hydroxy-1,4-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)diazenyl]phenyl benzenesulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification: FAT 93460/A
- Source and lot/batch No.of test material: EN-No.: 037098 A7
- Expiration date of the lot/batch: November, 2003

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability of the test substance in the solvent/vehicle: at least 24 hours in water, PEG 400, saline, FCA/NaCl-solution, and CMC-solution

OTHER SPECIFICS:
- Aggregate state at room temperature: Solid
- Colour: Yellow
- Purity: 23 % active ingredient

Method

Target gene:

Histidine requiring genes of Salmonella strains

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells:
The bacterial strains TA 1535, TA 98, and TA 100 were obtained from Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 1537 was obtained from BASF (D-67063 Ludwigshafen).

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 bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 µL ampicillin (25 µg/ml) 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 cultures were incubated in a shaking water bath for 8 hours at 37 °C.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : 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)
- method of preparation of S9 mix: Rats received daily applications of 80 mg/kg b.w. Phenobarbital i.p. dissolved in deionised water (Desitin; D-22335 Hamburg) and ß-Naphthoflavone orally dissolved in corn oil (Aldrich, D-89555 Steinheim) on three subsequent days. The livers were prepared 24 hours after the last treatment. After decapitation of the anaesthetised animals, the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate was diluted 1+3 in KCl and centrifuged at 9,000 g for 10 minutes at 4 °C. A stock of the supernatant containing the microsomes was frozen in ampoules and stored at -80 °C. Small numbers of the ampoules are kept at -20 °C for up to one week before use. The protein content was determined using an analysis kit of Bio-Rad Laboratories, D-80939 München (Bio-Rad protein assay, Catalogue No. 5000006). The protein concentration in the S9 preparation was 28.3 mg/ml (lot no. 271198) in the preexperiment and in experiment I, and 50.4 mg/ml (lot no. 181298) in experiment I and II.

- concentration or volume of S9 mix and S9 in the final culture medium: 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 in the cultures. The composition of the co-factor solution was chosen to yield the following concentrations in the S9 mix:
8mM MgCl2
33 mM KCl
5mM Glucose-6-phosphate
5mM 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.

Test concentrations with justification for top dose:

31; 92; 307; 920; 2300; and 4600 μg/plate (active ingredient)

Vehicle / solvent:

DMSO (purity >99 %, MERCK, D-64293 Darmstadt)

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:

Without metabolism: TA 1535 and TA 100

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

Without metabolism: TA 1537 and TA 98

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

With metabolic activation: TA 1535, TA 1537, TA 98, TA 100

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium, Plate incorporation assay.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
The bacterial cultures were incubated in a shaking water bath for 8 hours at 37 °C.

FOR GENE MUTATION:
- Method used: Agar

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

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98, and TA 100 or thrice in strains TA 1535 and TA 1537 (3, 4). 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 of whether the highest dose induced the criteria described above or not.

Evaluation criteria:

A test article is considered positive if either a dose related increase in the number of revertants or a biologically relevant increase for at least one test concentration is induced. A test article producing neither a dose related increase in the number of revertants nor a biologically relevant positive response at any one of the test points is considered nonmutagenic in this system. A biologically relevant response is described as follows: A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98, and TA 100 or thrice in strains TA 1535 and TA 1537. 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 criteria described above or not.

Statistics:

A statistical analysis of the data is not required.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Test article concentration per plate µg: 3, 9, 31, 92, 307, 920, 2300, 4600.
The plates with the test article showed normal background growth up to 4600 µg/plate in strain TA 98 and TA 100.

Any other information on results incl. tables

- No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

- The plates incubated with the test article showed normal background growth up to 4600 µg/plate with and without S9 mix in all strains used.

- Substantial and dose dependent increases in revertant colony numbers were observed following treatment with FAT 93460/A in all strains with and without metabolic activation in experiment I and II. The required threshold of twice (strains TA 98 and TA 100) and thrice (TA 1535 and TA 1537) the number of the corresponding solvent control was reached or exceeded at the following concentrations:

Strain	Experiment I (μg/plate)		Experiment II (μg/plate)
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	>307	> 307	>307	>307
TA 1537	>307	> 920	>307	> 92
TA 98	>920	> 31	> 92	> 31
TA 100	>920	>2300	>920	>920

Positive control substances showed a distinct increase in induced revertant colonies, thereby validating the test.

Applicant's summary and conclusion

Conclusions:

FAT 93460/A is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

A study was performed according to OECD test guideline 471 and EU Method B.13/14 to investigate the potential of FAT 93460/A to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The assay was performed 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: 31; 92; 307; 920; 2300; and 4600 μg/plate (active ingredient). No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. The plates incubated with the test article showed normal background growth up to 4600 μg/plate with and without S9 mix in all strains used. Substantial and dose dependent increases in revertant colony numbers were observed following treatment with FAT 93460/A in all strains with and without metabolic activation in experiment I and II. The required threshold of twice (strains TA 98 and TA 100) and trice (TA 1535 and TA 1537) the number of the corresponding solvent control was reached or exceeded at the concentrations. Based on the findings of the study, it can be concluded that the test article induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98, and TA 100. Therefore, FAT 93460/A is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.