Reaction mass of sodium potassium 7-amino-4-hydroxy-3,8-bis(2-{2-sulfonato-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)naphthalene-2-sulfonate, sodium potassium 7-amino-3-{2-[4-(ethenesulfonyl)-2-sulfonatophenyl]diazen-1-yl}-4-hydroxy-8-(2-{2-sulfonato-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)naphthalene-2-sulfonate, sodium potassium 7-amino-8-{2-[4-(ethenesulfonyl)-2-sulfonatophenyl]diazen-1-yl}-4-hydroxy-3-(2-{2-sulfonato-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)naphthalene-2-sulfonate and sodium potassium 7-amino-3,8-bis({2-[4-(ethenesulfonyl)-2-sulfonatophenyl]diazen-1-yl})-4-hydroxynaphthalene-2-sulfonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 17, 1998 to December 14, 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

"Kanpoan No. 287 ~ Environment Protection Agency"
"Eisei No. 127 -- Ministry of Health & Welfare"
"Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry"

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss GLP

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Batch No.: TVR50
Aggregate State at room temperature : solid
Colour: dark red / brown
Purity: 65%
Stability in solvent: 24 hours in water, saline, polyethylene glycol, CMC, vaseline, and FCA
Storage: room temperature
Expiration Date: September 01, 2004

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TVR50
- Expiration date of the lot/batch: September 01, 2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability of the test substance in the solvent/vehicle: 24 hours in water, saline, polyethylene glycol, CMC, vaseline, and FCA

Method

Target gene:

The Salmonella typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction from rat treated with phenobarbital and ß-Naphthoflavone.

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 33; 100; 333; 1000; 2500; and 5000 µg/plate
Concentration range in the main test (without metabolic activation): 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

Solvent: deionized water
The solvent was chosen because of its solubility properties.

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 metabolic activation- TA 1535, 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

Remarks:

Without metabolic activation- TA 1537, TA 98

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation- WP2 uvrA

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation- TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Details on test system and experimental conditions:

When summarised the mutations of the TA strains and the E. coli strain, 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
Escherichia coli
WP2 uvrA: trp; uvrA: base-pair substitutions and others
Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in RCC Cytotest Cell Research according to Ames et al.. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
The bacterial strains TA 1535, TA 98, and TA 100 were obtained from Dr. B.N. Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 1537 was obtained from BASF (D-67063 Ludwigshafen). The bacterial strain WP2 uvrA was obtained from Dr. Heinz Träger, Knoll AG, D-67008 Ludwigshafen.

Evaluation criteria:

A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.
A test article producing neither a reproducible and dose related increase in the number of revertants, nor a biologically relevant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.
A mutagenic response is described as follows:
A test article is considered mutagenic if in the strains TA 98, TA 100, and WP2 uvrA the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 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 statistical evaluation of data was required.

Results and discussion

Test results

Test results 1

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, and E. Coli WP2 uvrA

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

(5000 µg/plate), slight effects seen with TA 98 at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, and E. Coli WP2 uvrA

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

( 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Non mutagenic

Any other information on results incl. tables

The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.

Slight toxic effects, evident as a reduction in the number of revertants, occurred in strain TA 98 at 5000 µg/plate with S9 mix in experiment II.

In experiment II, the colony count of the negative control without S9 mix in strain TA 98 did not quite reach the lower limit of our historical range of negative control data. This effect is caused by statistical fluctuations of the rather low numbers of colonies in strain TA 98. Since the results of this study are based on the corresponding solvent control, which remained well within the range of our historical controls, this effect is judged irrelevant.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor 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.

Applicant's summary and conclusion

Conclusions:

FAT 40'571/A is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of FAT 40'571/A to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using theSalmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. This test was carried out according to OECD 471 and EU B.13 guidelines. 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 33; 100; 333; 1000; 2500; and 5000 µg/plate concentrations.

 

The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. Slight toxic effects, evident as a reduction in the number of revertants, occurred in strain TA 98 at 5000 µg/plate with S9 mix in experiment II.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 40'571/A at any dose level, neither in the presence nor 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 40'571/A is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.