Octasodium 2-(8-(4-chloro-6-(3-((4-chloro-6-(3,6-disulfonato-2-(1,5-disulfonatonaphthalen-2-ylazo)-1-hydroxynaphthalen-8-ylamino)-1,3,5-triazin-2-yl)aminomethyl)phenylamino)-1,3,5-triazin-2-ylamino)-3,6-disulfonato-1-hydroxynaphthalen-2-ylazo)naphthalene-1,5-disulfonate

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:

24 September 1991 to 11 October 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD test guidance in compliance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Octasodium 2-(8-(4-chloro-6-(3-((4-chloro-6-(3,6-disulfonato-2-(1,5-disulfonatonaphthalen-2-ylazo)-1-hydroxynaphthalen-8-ylamino)-1,3,5-triazin-2-yl)aminomethyl)phenylamino)-1,3,5-triazin-2-ylamino)-3,6-disulfonato-1-hydroxynaphthalen-2-ylazo)naphthalene-1,5-disulfonate

EC Number:

413-550-5

EC Name:

Octasodium 2-(8-(4-chloro-6-(3-((4-chloro-6-(3,6-disulfonato-2-(1,5-disulfonatonaphthalen-2-ylazo)-1-hydroxynaphthalen-8-ylamino)-1,3,5-triazin-2-yl)aminomethyl)phenylamino)-1,3,5-triazin-2-ylamino)-3,6-disulfonato-1-hydroxynaphthalen-2-ylazo)naphthalene-1,5-disulfonate

Cas Number:

142068-96-0

Molecular formula:

Hill formula: C53H28Cl2N14Na8O26S8

IUPAC Name:

octasodium 5-[(4-chloro-6-{[4-({[4-chloro-6-({7-[2-(1,5-disulfonatonaphthalen-2-yl)diazen-1-yl]-8-hydroxy-3,6-disulfonatonaphthalen-1-yl}amino)-1,3,5-triazin-2-yl]amino}methyl)phenyl]amino}-1,3,5-triazin-2-yl)amino]-3-[2-(1,5-disulfonatonaphthalen-2-yl)diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate

Details on test material:

A Test Sample of Substance H112339 (Batch Ref: NBY 405/74) was supplied by ICI Specialties, as a dark brown powder. It was assigned the CTL reference number Y07719/001. From the information supplied by the Sponsor, the sample was stable under normal storage conditions and under the test conditions used in this study. In all cases where the concentration of the Test Sample is quoted, this concentration (eg u.g/plate) refers to the weight of sample as supplied.

Method

Target gene:

Not applicable.

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:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

200 - 6983.2 μg per plate (The maximum dose level of 6983.2 μg/plate is approximately equal to 5000 μg of the main component of Substance H112339 per plate with consideration given to the water impurity).

Vehicle / solvent:

Sterile deionised water was used as the solvent/diluent for the test compound, and as the negative control

Controls

Untreated negative controls:

yes

Remarks:

Sterile deionised water

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulphoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: x4 substances

Remarks:

+ve control substances listed in any other information

Details on test system and experimental conditions:

Summary of Methodology: The mutagenicity assays were conducted using the Salmonella bacterial mutation assay as described by Maron and Ames (19S3), as updated by the United Kingdom Environmental Mutagen Society's sub-committee on Guidelines for Mutagenicity Testing (Gatehouse et al, 1990).

The Test Sample of Substance was initially assayed using the standard plate incorporation protocol over a dose range of 200 - 6983.2μg per plate, bath in the presence and absence of a liver S9-mix prepared from AROCLOR 1254-induced Charles River CD [Crl:CD(SD)BR] rats. The four Salmonella tester strains (TA1535, TA1537, TA9S and TA100) and two E.col i strains (WP2P and WP2P uvrA) used in this assay have been fully described in the literature (Ames et al 1975, and references therein; Venitt and Crafton~Steigh, 1979). The compound was subsequently re-tested in all six strains over the same dose range: the +S9 phase of this second assay was conducted using the Pre-Incubation protocol. In light of the slight effects observed in this second +S9 experiment, the compound was re-tested in strains WP2P and WPZP uyrA (+S9 only), again using a Pre-Incubation protocol. 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/S9 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) experiment is achieved when:
a) There is no statistically significant dose-related increase i- the mean number of revertant colonies per plate observed for the *5t compound; and
b) in the absence of any such dose response, no increase in ceiony 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/S9 combination, then the observed effect(s) must be consistently reproducible.

All derived calculations (ie mean colony count/plate; standard deviation, etc) shown in the results tables were carried out by computer.

NB In the results tables some colony counts are replaced with the letter 'C This denotes a contaminated plate: counts from such plates are not included in the calculations of mean colony count and standard deviation

Statistics:

An initial assessment of statistical significance was carried out using a one-tailed Student's t-test (Ehrenberg 1984). The corresponding probability for each dose leveT was derived by computer using the appropriate degrees of freedom. Values of p<0,01 are treated as significant, with values of 0.01

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:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test compound, together with appropriate positive and negative controls, was examined in at least two independent experiments both in the presence and 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 H112339 gave a negative, ie non-mutagenic response in S-typhimurium strains TA1535, TA1537, TA98 and TA100 and in E.coli strains WP2P and WP2P uvrA in both the presence and absence of S9.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the conditions of this assay, Substance H112339 gave a negative, ie non-mutagenic, response with S-typhimurium strains TA1535, TA1537, TA98 and TA100 and with E.coli strains WP2P and WP2P uvrA in the presence and 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 H112339 gave a negative, ie non-mutagenic response in S-typhimurium strains TA1535, TA1537, TA98 and TA100 and in E.coli strains WP2P and WP2P uvrA in hath the presence and absence of S9

Executive summary:

Study conducted to OECD test guidelines 471 and 472 in compliance with GLP.

The substance is non mutagenic with and without metabolic activation.