Reaction mass of 3-(2-{3-cyanato-5-[2-(3-cyanatoaryl)heteropolycycle-5-yl]aryl}heteropolycycle-5-yl)aryl cyanate and heteropolycycle -2,5-diyldi-3,1-arylene dicyanate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic potential was determined in a GLP-compliant study in accordance with OECD no. 471, EU methods B.13/B.14 and the corresponding Japanese testing guidelines. It is concluded that LZ1554 showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

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:

22 November 2011 to 6 January 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Qualifier:

according to guideline

Guideline:

other: Official notice of MHLW, METI and MOE (31 March 2011) YAKUSHOKUHATSU 0331 No 7 SEIKYOKU No 5 KANPOKIHATSU No 110331009

Qualifier:

according to guideline

Guideline:

other: Official Notice of J MOL (8 February 1999)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine dependency S. typhimurium
Tryptophan dependency Escherichia coli

Species / strain / cell type:

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

Additional strain / cell type characteristics:

nitroreductase deficient

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver

Test concentrations with justification for top dose:

Test 1: 5, 15, 50, 150, 500, 1500, 5000 µg per plate
Test 2: 50, 150, 500, 1500, 5000 µg per plate

Vehicle / solvent:

The solubility of the test substance was assessed at 50 mg/mL in both DMF and dimethyl sulphoxide (DMSO). It was found to be soluble in both solvents. DMSO (ACS spectrophotometric grade) was selected as the vehicle for this study since it is less toxic to the test system.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

used for TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

used for TA100 and TA1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

used for TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

used for E. coli WP2 uvrA pKM101

Details on test system and experimental conditions:

Test 1
Aliquots of 0.1 mL of the test substance solutions (seven concentrations up to 5000 μg/plate), positive control or vehicle control were placed in glass tubes. The vehicle control was DMSO. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was
individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37°C for ca 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter. Some plates were scored manually because of the presence of precipitate.

Test 2
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes with shaking before the addition of the agar overlay. The maximum concentration chosen was again 5000 μg/plate, but only five concentrations were used.

Evaluation criteria:

If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed. If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.

Statistics:

If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis.

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:

cytotoxicity

Remarks:

1st & 2nd test - at 5000 µg/plate toxicity and precipitation were observed

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitating concentration is 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

First test - No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to LZ1554 at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

Second test - No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to LZ1554 at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test substance formulation.

The viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded E09/mL in all cases, and therefore met the acceptance criteria.

The mean revertant colony counts for the vehicle controls were within or close to the current historical control range of the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that LZ1554 showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Executive summary:

The mutagenic potential was determined in a GLP-compliant study in accordance with OECD no. 471, EU methods B.13/B.14 and the corresponding Japanese testing guidelines. It is concluded that LZ1554 showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the data available the substance is not classified or labeled according to Directive 67/548/EEC (DSD) or Regulation 1272/2008/EC (CLP).