Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Ames Test: potent and definite mutagen in the Ames Test; strongly positive in TA 98 and positive in TA 100 Mouse bone marrow micronucleus test, i.p.: negative
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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP Study, only 4 Salmonella strains used
Principles of method if other than guideline:
Salmonella/microsome test, plate incorporation assay, as described by Ames et al., (1973a, 1975) and Maron and Ames (1983); only 4 Salmonella strains used
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
mutant histidine gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix was made from the livers of male Sprague Dawley rats, which received a single intraperitoneal injection of 500 mg/kg bw Aroclor 1254, dissolved in corn oil, 5 days prior to sacrifice. The S9 mix comprised 30 % S9 fraction.
Test concentrations with justification for top dose:
plate incorporation assay:
0, 8, 40, 200, 1000, and 5000 µg/plate with and without S9 mix (first and second trial)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
The highest dose of Delta-R-Base was applied as suspension.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
Na-azide (only TA 1535), nitrofurantoin (only TA 100), 4-nitro-1,2-phenylene diamine (TA 1537 and TA 98), 2-aminoanthracene
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
Statistics:
no statistics performed; evaluation based on criteria mentioned above
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
On TA 100 and TA 98 a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Positive response was found only with S9 mix at 40 µg/plate (TA 100) and 8 µg/plate (TA 98).
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 5000 µg/plate the substance had a weak, strain specific bacteriotoxic effect, so that dose could nevertheless be used for assessment purposes. Substance precipitation occurred at 5000 µg/plate.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Two of the five strains (TA 98 and TA 100) in the plate incorporation test with S9 mix showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. For TA 98 this increase was even higher than that of the positive control 2-aminoanthracene. TA 98 counts for frameshift effects and TA 100 for base-pair substitution.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Summary and mean values with S9 mix

Table and group µg/plate

Strain

TA 1535

Strain

TA 100

Strain

TA 1535

Strain

TA 98

+ S9 (30 %)

 

plates 1-4 / 5-8

0 (Ethanol)

20 / 14

147 / 146

10 / 9

53 / 53

8

20 /15

188 /143

14 / 14

242 / 291

40

21 /16

257 / 292

23 / 20

521 / 485

200

24 /13

379 / 413

17 / 19

502 / 638

1000

18 / 17

426 / 370

26 / 24

499 / 601

5000

20 / 16

444 / 432

23 / 25

P / P

2-AA (pos. control)

204 / 84

758 / 902

81 / 81

437 / 381

P = precipitation
numbers in bold show values which led to a statistical significance in the respective quotients

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation
Executive summary:

The test substance was investigated in the Ames Test using Salmonella typhimurium strains TA 1535, TA 100, TA 1537, and TA 98 for point mutagenic effects in doses of 8 up to 5000 µg/plate. At 5000 µg/plate a weak, strain specific bacteriotoxic effect and precipitation of the test substance appeared. Clear evidence of mutagenic activity of the test substance was seen in cultures with S9-mix at doses of >= 8 µg/plate for TA 98 and at >= 40 µg/plate for TA 100. For TA 98 the dose-dependent increase reached higher mutant frequencies than the positive control 2-aminoanthacene. TA 98 and TA 100 cover different mutagenic effects, i.e. frameshift effects and base-pair substitution, respectively. Thus, the test substance should be regarded as a potent and definite mutagen under the conditions of the Salmonella/microsome assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vitro:

The test substance was investigated in the Ames Test using Salmonella typhimurium strains TA 1535, TA 100, TA 1537, and TA 98 for point mutagenic effects in doses of 8 up to 5000 µg/plate. At 5000 µg/plate a weak, strain specific bacteriotoxic effect and precipitation of the test substance appeared. Clear evidence of mutagenic activity of the test substance was seen in cultures with S9-mix at doses of >= 8 µg/plate for TA 98 and at >= 40 µg/plate for TA 100. For TA 98 the dose-dependent increase reached higher mutant frequencies than the positive control 2-aminoanthacene. TA 98 and TA 100 cover different mutagenic effects, i.e. frameshift effects and base-pair substitution, respectively. Thus, the test substance should be regarded as a potent and definite mutagen under the conditions of the Salmonella/microsome assay.

A micronucleus test on male and female NMRI mice was performed according to OECD TG 474 to investigate potential clastogenic effects of the test item. Groups of 5 mice/sex received a single intraperitoneal injection of either the test substance (750 mg/kg bw), the vehicle (corn oil), or the positive control substance cyclophosphamide (20 mg/kg bw). The femoral marrow of the groups treated with the test item was prepared 16, 24 and 48 hours after administration.

All animals treated with the test item showed symptoms of toxicity after treatment, as apathy and roughened fur. A weak altered ratio between poly- and normochromatic erythrocytes in the 48 h treated group pointed to exposure of the bone marrow. No indications of clastogenic effects of the test item were found. Thus, the test item was not clastogenic under the conditions of this test.

Justification for classification or non-classification

The available data for the test item indicate that the substance is no inducer of chromosomal aberrations or aneugenic effects in somatic mammalian cells in vivo. In bacteria, however, a point mutagenic effect was seen in two Salmonella strains. The two affected strains TA 98 and TA 100 cover different types of gene-mutagenic effects, i.e. frameshift mutations and base-pair substitutions, respectively. Therefore and because the mutagenic effect, especially in TA 98, was strong and dose-dependent, the test material should be regarded as a potent and definite mutagen in bacteria.

Based on the available information (a strongly positive Ames Test and a negative mouse micronucleus test in vivo) a classification according to EU Regulation 1272/2008 is not adequate.