Trisodium hydrogen [2-[[α-[[3-[[4-chloro-6-[ethyl[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-2-hydroxy-5-sulphophenyl]azo]benzyl]azo]-4-sulphobenzoato(6-)]cuprate(4-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance did not induce mutation in Salmonella typhimurium TA98, TA100, TAl535 and TA1537 strains and one Escherichia coli WP2 uvrA, in the presence and absence of a rat liver metabolic activation system (S-9).

Link to relevant study records

Reference

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:

From September 19th to October 13rd, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test conducted according to internationally accepted testing guidelines.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1993

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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 Salmonella typhimurium tester strains were originally obtained from the UK NCTC, and the Escherichia coli strain from the Cancer Research Unit, University of York. For all assays, bacteria were cultured for 10 hours at 37 °C in nutrient broth (containing ampicillin for strains TA98 and TA100). Incubation was carried out in a shaking incubator. Bacteria were taken from vials of frozen cultures, which had been checked for strain characteristics of histidine or tryptophan dependence, rfa character (Salmonella strains) and presence (strains TA98 and TA100) or absence (strains TAl135, TA1537 and Escherichia coli) of the pKM101 ampicillin resistance factor.
All experimentation commenced within 2 hours of the end of the period of incubation.

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

Range-finder Experiment and Mutation Experiment 1: 8, 40, 200, 1000 and 5000 µg/plate
Mutation Experiment 2: 1000, 2000, 3000, 4000 and 5000 µg/plate

Vehicle / solvent:

- Vehicle: sterile purified water.

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Remarks:

Without S9: 2NF for TA98, NaN3 for TA100 and TA 1535, AAC for TA1537, NQO for WP2uvrA. With S9: AAN for all strains

Details on test system and experimental conditions:

RANGE FINDING TEST
The test item was tested in strain TA100. Triplicate plates without and with S-9 mix were used. Negative (solvent) and positive controls were included in quintuplicate and triplicate respectively, without and with S-9 mix. These platings were achieved by the following sequence of additions to 2.5 ml molten agar at 46 °C: 0.1 ml bacterial culture, 0.1 ml test article solution or control and 0.5 ml 10 % S-9 mix or buffer solution followed by rapid mixing and pouring on to Minimal Davis agar plates. When set, the plates were inverted and incubated at 37 °C in the dark for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.

MUTATION EXPERIMENTS
The test item was tested in four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TAl537) and one strain of Escherichia coli (WP2 uvrA), in two separate experiments using triplicate plates without and with S-9.
Negative (solvent) controls were included in each assay, in quintuplicate without and with S-9. In each experiment, bacterial strains were treated with diagnostic mutagens in triplicate in the absence of S-9. The activity of the S-9 mix used in each experiment was confirmed by AAN treatments (again in triplicate) of at least one strain in the presence of S-9.

As the results of the first experiment were negative, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step, where the quantities of test article or control solution, bacteria and S-9 mix, were mixed together and incubated for 20 minutes at 37 °C, before the addition of 2.5 ml molten agar at 46 °C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. ln this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.

COLONY COUNTING
Colonies were counted electronically using a Seescan Colony Counter or manually, where physical effects (e.g split agar) interfered with the accuracy of the automated counter. All dose plates treated at concentrations of 1000 µg/plate or above were scored manually due to the dark colour of the test article.
For the Experiment 2 treatments, all solvent and dose plates were counted manually for consistency in counting methods between the treatment and negative control plates.
The background bacterial lawn of each test plate was inspected for signs of toxicity.

ACCEPTANCE CRITERIA
The assay was considered valid if the following criteria were met:
1) the mean negative control counts fell within the normal ranges
2) the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9 preparation
3) no more than 5 % of the plates were lost through contamination or some other unforeseen event.

METABOLIC ACTIVATION SYSTEM
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor 1254.
Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).

Evaluation criteria:

The test article was considered to be mutagenic if:
1) the assay was valid (acceptance criteria)
2) Dunnett‘s test gave a significant response (p ≤ 0.01) and the data set showed a significant dose correlation
3) the positive responses were reproducible.

Statistics:

The m-statistic was calculated to check that the data were Poisson-distributed and Dunnett's test was used to compare the counts of each dose with the control.
The presence or otherwise of a dose response was checked by linear regression analysis.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MUTATIONS
Only Range-finder experiment treatments of strain TA100, in the presence of S-9, resulted in a statistically significant increase in revertant numbers (when the data were analysed at the 1 % level using Dunnett’s test). The increase was very small in magnitude and provided no clear evidence of a dose-relationship. Furthermore, the increase could not be deemed to be reproducible when considered alongside analogous treatments in Experiment 1 and Experiment 2 where no statistically significant increase occurred.
None of the test treatments were therefore considered to have provided any clear evidence of test item mutagenic activity, the increase in revertant numbers that did occur being attributed to chance.

TOXICITY AND PRECIPITATIN
Experiment 1 treatments were performed using the same dose range as employed for the Range-finder experiment. Following these treatments, no clear evidence of toxicity was observed.
Experiment 2 treatments retained the same maximum test dose, but utilised a narrowed dose range in order to more closely investigate those concentrations of the test item most likely to demonstrate a mutagenic response. In addition, all treatments in the presence of S-9 employed a pre-incubation step. Following these treatments, again no evidence of toxicity was observed.
The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

CONTROLS
Mean solvent control counts fell within the normal historical ranges; the positive control chemicals all induced large increases in revertant numbers in the appropriate strains, and that less than 5 % of plates were lost, leaving adequate numbers of plates at all treatments. The study was accepted as valid.

RANGE-FINDING/SCREENING STUDIES
An initial toxicity range-finder experiment was carried out in strain TA100 onl.
Following these treatments, no evidence of toxicity (which would normally be indicated by a thinning of the bacterial background lawn and/or a marked reduction in revertant numbers) was observed. In order to assess the reproducibility of significant increases in revertant numbers observed following the Range-finder experiment, strain TA100 was treated alongside the other tester strains in Experiment 1.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The substance did not induce mutation under test conditions.

Executive summary:

The substance was assayed for mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2 uvrA) of Escherichia coli, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post~mitochondrial fraction (S-9), in two separate experiments.

An initial toxicity Range-finder experiment was carried out in strain TA100 only, using final concentrations of test item at 8, 40, 200, 1000 and 5000 µg/plate, plus negative (solvent) and positive controls. Following these treatments, no clear evidence of toxicity was observed. ln order to assess the reproducibility of a significant increase in revertant numbers observed following the Range-finder experiment, strain TA100 was treated alongside the other tester strains in Experiment l. Experiment 1 treatments were performed using the same dose range employed in the Range-finder experiment. Following these Experiment 1 treatments, no clear evidence of toxicity was observed.

Experiment 2 treatments retained the same maximum test dose, but utilized a narrow dose range in order to more closely investigate those concentrations of test item considered most likely to demonstrate a mutagenic response (1000-5000 µg/plate). In addition, all treatments in the presence of S-9 employed a pre-incubation step, and in this way it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system.

Following these Experiment 2 treatments, once again, no clear evidence of toxicity was observed.

Negative (solvent) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments.

Only Range-finder experiment treatments of strain TA100, in the presence of S-9, resulted in a statistically significant increase in revertant numbers (when the data were analysed at the 1 % level using Dunnett’s test). The increase was very small in magnitude and

provided no clear evidence of a dose-relationship. Furthermore, the increase could not be deemed to be reproducible when considered alongside analogous treatments in Experiment 1 and Experiment 2, where no statistically significant increase occurred.

None of the test treatments were therefore considered to have provided any clear evidence of test substance mutagenic activity, the increase in revertant numbers that did occur being attributed to chance.

Conclusion

It was concluded that the substance did not induce mutation in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TAl535 and TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2 uvrA), under the conditions employed in this assay system, which included treatments up to a maximum test concentration of 5000 µg/plate, in the presence and absence of a rat liver metabolic activation system (S-9).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The test substance did not show any reasons of concern in the test performed.

In conclusion, the substance is not classified for genetic toxicity according to the CLP Regulation (EC 1272/2008).