Pentasodium bis[5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-5-nitrophenyl)azo]naphthalene-2,7-disulphonato(4-)]chromate(5-)

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:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

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:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Principles of method if other than guideline:

None

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Pentasodium bis[5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-5-nitrophenyl)azo]naphthalene-2,7-disulphonato(4-)]chromate(5-)

EC Number:

279-317-3

EC Name:

Pentasodium bis[5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-5-nitrophenyl)azo]naphthalene-2,7-disulphonato(4-)]chromate(5-)

Cas Number:

79828-43-6

Molecular formula:

C38H18Cl2CrN16O20S4.5Na

IUPAC Name:

Pentasodium bis[5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-5-nitrophenyl)azo]naphthalene-2,7-disulphonato(4-)]chromate(5-)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

None

Method

Target gene:

Salmonella typhimurium histidine (his) reversion system

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: his(+), uvrB(-), rfa(+), R factor(+)for TA 98 and TA 100; R factor(-)for TA 1535 and TA 1537

Species / strain 2

Species / strain / cell type:

E. coli, other: E.coli WP2P and WP2PuvrA

Additional strain / cell type characteristics:

other: uvrA mutation

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix (Rat liver enzymes)

Test concentrations with justification for top dose:

100 - 5000 µg/plate

Vehicle / solvent:

water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

for all strains with S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100, TA 1535 without S9

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Acridine Mutagen ICR191

Remarks:

TA 1537 without S9

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin HCl

Remarks:

TA 98 without S9

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

E. coli WP2P without S9

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

E. coli WP2P uvrA· without S9

Details on test system and experimental conditions:

A solution of the test compound (as supplied) was prepared in sterile (filtered: Milli-Q/RO) water to yield a w/v concentration of 50.00 mg/ml.
This was then diluted with water to give additional solutions of 25 mg/ml, 10 mg/ml, 5.0 mg/ml, 2.0 mg/ml and 1.0 mg/ml. Fresh stock solutions and dilutions were prepared as necessary for each experiment.
Water was also included as the negative (solvent) control.

The mutagenicity assays were conducted using the Salmonella bacterial mutation assay as described by Maron and Ames (1983), as updated by the United Kingdom Environmental Mutagen Society's sub-committee on Guidelines for Mutagenicity Testing (Gatehouse et al, 1990) (see Appendix A). The four Salmonella tester strains (TA1535, TA1537, TA98 and TA100) and two E.coli strains (WP2P and WP2PuvrA) used in this assay have been fully described in the literature (Ames et al 1975, and references therein; Venitt and Crofton-Sleigh, 1979).
The sample of Substance S70767 was assayed twice using the standard plate incorporation protocol over a dose range of 5000 - 100 µg per plate using all six strains (in a total of three separate experimental runs), both in the presence and absence of a liver S9-mix prepared from Phenobarbital/ beta-Naphthaflavone-induced Alderley Park (Alpk:APfSD) rats.
The incubation period for each experiment was 3 days (at 37°C).

For each experiment, positive control compounds were tested to validate the bacterial strains and to confirm the activity of each batch of S9-mix used.
Revertant colonies were counted using an automated electronic colony counter (AMS 40-10 Image Analyser fitted with appropriate software, Analytical Measuring Systems Ltd).

Evaluation criteria:

Test data from individual experiments are considered valid if:
a) the concurrent solvent control data are acceptable;
b) the positive control data show unequivocal positive responses;
c) at least the lowest test compound dose shows no evidence of toxicity, and at least three test doses show no significant toxicity (ie significant loss of background growth and/or reductions in colony numbers).
Failure of one or more tester strain/59 combinations does not invalidate the data for the remainder of a concurrent experiment.

A positive response in a (valid) individual experiment is achieved when one or both of the following criteria are met:
a) a statistically significant dose-related increase in the mean number of revertant colonies is obtained;
b) a two-fold or greater increase in the mean number of revertant colonies (over that observed for the concurrent solvent control plates) which is
statistically significant, is observed at at least one dose level.

A negative result in a (valid) individual experiment is achieved when:
a) There is no statistically significant dose-related increase in the mean number of revertant colonies per plate observed for the test compound; and
b) in the absence of any such dose response, no increase in colony numbers is observed (at any test dose) which exceeds 2x the concurrent solvent control.

For a positive response in an individual experiment to be considered indicative of an unequivocal positive, ie mutagenic, result for that strain/59 combination, then the observed effect(s) must be consistently reproducible.

Statistics:

All derived calculations (ie mean colony count/plate; standard deviation, etc) shown in the results tables were carried out by computer.
Counts from contaminated plates are not included in these calculations.

An assessment of statistical significance was carried out using a one-tailed Student's t-test (Ehrenberg 1984). The corresponding probability for each dose level was derived by computer using the appropriate degrees of freedom. Values of p<0.01 are treated as significant, with values of 0.01<=p<0.05 being indicative of a possible effect.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

but tested up to limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

but tested up to limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

but tested up to limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In two separate assays, the compound induced significant, reproducible increases in the observed number of revertant colonies in strains TA1535, TA1537, TA98, TA100 and WP2P both in the presence or absence of an auxiliary metabolising system (S9). Limited activity was observed in strain WP2PuvrA both in the presence and absence of S9.

The positive controls for each experiment induced the expected responses, indicating the strains were responding satisfactorily in each case.

Applicant's summary and conclusion

Conclusions:

The test substance gave a positive, ie mutagenic response in S.typhimurium strains TA1535, TA1537, TA98 and TA100 and in E.coli strain WP2P in both the presence and absence of S9. Limited activity was observed in E.coli strain WP2PuvrA in both the presence and absence of S9.

Executive summary:

A study was performed to determine the mutagenecity of Substance S70767 according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) and OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay). Substance S70767 has been evaluated in a bacterial mutagenicity assay (Gatehouse et al, 1990: based on Maron and Ames (1983)) using four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and two strains of Escherichia coli (WP2P and WP2PuvrA), following protocols complying with OECD Guideline Numbers 471 and 472 (OECD, 1983a and 1983b) and with the United Kingdom Department of Health Guidelines (DoH, 1989).

In two separate experiments, the compound induced significant, reproducible increases in the observed numbers of revertant colonies in all of the tester strains used, both in the presence or absence of an auxiliary metabolising system (S9). In each experiment, the positive controls responded as expected indicating that the assay was performing satisfactorily. Under the conditions of this assay, Substance S70767 therefore gave a positive, ie mutagenic response in S.typhimurium strains TA1535, TA1537, TA98 and TA100 and in E.coli strain WP2P in both the presence and absence of S9. Limited activity was observed in E.coli strain WP2PuvrA in both the presence and absence of S9.