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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Genetic toxicity in vitro - Gene Mutation in Bacteria (Bacterial Reverse Mutation Assay/Ames): negative with and without metabolic activation (equivalent to OECD471) - In vitro chromosomal aberration test (in human lymphocytes): negative with and without metabolic activation (similar to OECD TG 473) - Gene Mutation in Mammalian Cells (L5178Y TK+/- Mouse Lymphoma Mutagenesis Assay): negative with and without metabolic activation (according to OECD476)
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:
The experimental phase of the study was conducted between 8 December 1994 and 16 January 1995.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to OECD guidelins and in compliance with GLP.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
No certificate included in report
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell lines (if applicable):
Not applicable.
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-1254 induced rat liver, S9
Species / strain:
S. typhimurium TA 1538
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-1254 induced rat liver, S9
Species / strain:
E. coli WP2 uvr A
Details on mammalian cell lines (if applicable):
Not applicable.
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-1254 induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 5, 50, 500, 5000 µg/plate

Mutation Test Concentrations:
Experiment 1: 0, 39.036, 78.125, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate.
Experiment 2: 0, 39.036, 78.125, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate

Vehicle:
Vehicle: Dimethyl sulphoxide

Justification for choice of solvent/vehicle:
Prior to commencing testing, the solubility of the test substance was assessed at 50 mg/ml in water in which it was not fully miscible and dimethyl sulphoxide in which it was fully miscible. Dimethyl sulphoxide was therefore chosen as the solvent for this study.

Negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 1 µg/plate
Remarks:
With S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535 and TA1537
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98 and TA1538
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene, 0.5 µg/plate
Remarks:
With S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 10 µg/plate
Remarks:
With S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-ethyl-N-nitro-N-nitrosoguanidine, 3 µg/plate
Remarks:
without S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: N-ethyl-N-nitro-N-nitrosoguanidine, 5 µg/plate
Remarks:
Without S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix

Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate
Negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Nitrofluorene, 2 µg/plate
Remarks:
without S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1538
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Nitrofluorene, 1 µg/plate
Negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: N-ethyl-N-nitro-N-nitrosoguanidine, 2µg/plate
Remarks:
Without S9 mix
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation) Experiments 1 and 2

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.
Evaluation criteria:
Acceptance Criteria:
The mean number of revertant colonies for all treatment groups is compared with those obtained for solvent control groups. The mutagenic activity of a test substance is assessed by applying the following criteria:
(a) If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S-9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(b) If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
(c) If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in paragraphs (a) and (b), the following approach is taken in order to resolve the issue of the substance's mutagenic activity in this test system.

(i) Repeat tests may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of a narrower dose range than that already tested; the use of different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies paragraph (a) the substance is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(ii) If no clear "positive" response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al (1989).
Statistics:
Standard deviation
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
The revertant colony counts for Salicynalva obtained in the preliminary toxicity test was toxic at 5000 µg/plate towards all the tester strains in the presence of S-9 mix and towards all strains except TA 1535 and WP2 uvrA in the absence of S-9 mix. Therefore, 5000 µg/plate was chosen as the top dose level in the first mutation test but a total of eight dose levels were tested to ensure that sufficient non-toxic dose levels were obtained for all the tester strains. Toxicity was shown towards all the tester strains, although the degree of toxicity was strain dependent. The lowest dose level at which toxicity was observed was 625 µg/plate and toxicity was observed towards all the tester strains at 5000 µg/plate. At least four non-toxic dose levels were obtained for all the strains so it was decided to use the same dose levels in the second mutation test.

Toxicity towards the tester strains was again observed. This followed the same pattern as that observed in the first mutation test. No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment with Salicynalva at any dose level, in the presence or absence of S-9 mix, in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

REFERENCES

AMES, B.N., MCCANN, J., and YAMASAKI, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutation Research, 31, 347.

 

EEC (1993) EEC Methods for Determination of Toxicity, Annex to Directive 92/69/EEC, (01 No. L383A, 29. 1 2.92), Part B, Method B. 14. Other effects - Mutagenicity: Salmonella typhimurium - Reverse Mutation Assay.

 

EEC (1993) EEC Methods for Determination of Toxicity, Annex to Directive 92/69/EEC, (01 No. L383A, 29. 1 2.92), Part B, Method B.13. Other effects - Mutagenicity: Escherichia coli – Reverse Mutation Assay.

 

EPA (1984) US Environmental Protection Agency, Method: HG-Gene Muta - S. typhimurium: TheSalmonella typhimurium reverse mutation assay, 1 984.

 

GREEN, M.H.L. (1984) Mutagen testing using trp+ reversion in Escherichia coli in: KILBEY, B.1., LEGATOR, M., NICHOLS, W. and RAMEL, C. (Eds). Handbook of Mutagenicity Test Procedures. Second edition, p. 1 6 1 . Elsevier Science Publishers BV, Amsterdam.

 

JMAFF (1985) Japan, Ministry of Agriculture, Forestry and Fisheries, Notification of Director General, Agricultural Production Bureau, NohSan No. 4200, 28 January 1985.

 

JMITI (1986 and 1987) Japan, Joint Directives of the EPA, MOHW and MITI: Kampo-gyo No. 700 (EPA), Yakuhatsu No. 1 039 (MOHW), 6 1 Kikyoko No. 1 0 1 4 (MITI), 5 December 1 986; Kampo-gyo No. 237 (EPA), Yakuhatsu No. 306 (MOHW), 62 Kikyoku No. 303 (MITI), 31 March 1 987.

 

JMOHW (1989) Japan, Ministry of Health and Welfare, Notification Yakushin 1 No. 24, 11 September 1989, Guidelines for Toxicity Studies of Drugs, 4 I, Bacterial Reverse Mutation Test.

 

JMOL (1987) Japan, Ministry of Labour, Guidebook of Mutagenicity Tests, Published 16 June 1987.

 

MAHON, G.A.T., GREEN, M.H.L., MIDDLETON, B., MITCHELL, LDE G., ROBINSON, W.D. and TWEATS, D.J. (1989) Analysis of data from microbial colony assay in: KIRKLAND, DJ. (Ed.) UKEMS Subcommittee on Guidelines for Mutagenicity Testing. Report, Part Ill. Statistical Evaluation of Mutagenicity Test Data, p.26. Cambridge University Press, Cambridge.

 

MARON, D.M. and AMES, B.N. (1983) Revised methods for the Salmonella mutagenicity test. Mutation Research, 113, 173.

OECD (1983) OECD Guidelines for Testing of Chemicals No. 47 1 : Genetic Toxicology: Salmonella typhimurium, Reverse Mutation Assay, 26 May 1983.

 

OECD (1983) OECD Guidelines for Testing of Chemicals No. 472: Genetic Toxicology:Escherichia coli, Reverse Mutation Assay, 26 May 1983.
Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that, when tested in dimethyl sulphoxide, Salicynalva was not mutagenic in this bacterial system.
Executive summary:

In this in vitro assessment of the mutagenic potential of Salicynalva, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and a tryptophan dependent mutant of Escherichia coli (WP2 uvrA) were exposed to the test substance, diluted in dimethyl sulphoxide which was also used as a negative control. Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats. In the preliminary toxicity test, with dose levels of up to 5000 p-g/plate, toxicity was observed at the top dose level towards all the tester strains in the presence of S-9 mix and all the strains except TA 1535 and WP2 uvrA in the absence of S-9 mix. A top dose level of 5000 p-g/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 2500, 1250, 625, 312.5, 156.25, 78.125 and 39.063 µg/plate.

No evidence of mutagenic activity was seen at any dose level of Salicynalva in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, when tested in dimethyl sulphoxide, Salicynalva was not mutagenic in this bacterial system.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Ames test: In this in vitro assessment of the mutagenic potential of Salicynalva, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and a tryptophan dependent mutant of Escherichia coli (WP2 uvrA) were exposed to the test substance, diluted in dimethyl sulphoxide which was also used as a negative control. Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats. In the preliminary toxicity test, with dose levels of up to 5000 p-g/plate, toxicity was observed at the top dose level towards all the tester strains in the presence of S-9 mix and all the strains except TA 1535 and WP2 uvrA in the absence of S-9 mix. A top dose level of 5000 p-g/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 2500, 1250, 625, 312.5, 156.25, 78.125 and 39.063 µg/plate. No evidence of mutagenic activity was seen at any dose level of Salicynalva in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that, when tested in dimethyl sulphoxide, Salicynalva was not mutagenic in this bacterial system.

Chromosomal aberrations: A study was performed to assess the ability of Salicynalva to induce chromosomal aberrations in human lymphocytes cultured in vitro. Cultured human lymphocytes, stimulated to divide by addition of phytohaemagglutainin, were exposed to the test substance both in the presence and absence of S9 mix derived from rat livers. Solvent and positive control cultures were also prepared.After the appropriate treatment time, cell division was arrested using colchicines, the cells harvested and slides prepared, so that metaphase figures could be examined for chromosomal damage. In order to assess the toxicity of Salicynalva to cultured human lymphocytes, the mitotic index was calculated for all cultures treated with the test substance and the solvent control.On the basis of these data, the following concentrations were selected for metaphase analysis:

First test: Without S9mix; 18 hour harvest: 75, 63.5 and 31.25 µg/mL; With S9mix; 18 hour harvest: 187.5, 125 and 62.5 µg/mL. Second test: Without S9mix; 18 hour harvest: 75, 31.25 and 15.6 µg/mL; With S9 mix; 18 hour harvest: 250, 187.5 and 125 µg/mL

Without S9mix; 32 hour harvest: 75 µg/mL; With S9mix; 32 hour harvest: 250 µg/mL. There were no statistically significant increases in the number of aberrant cells, in the presence of S9 mix, at the 18 or 32 hour harvest. In the absence of S9-mix, in the second test at 18 hour harvest, there was a statistically significant increase in the number of aberrant cells at the lowest concentration, 15.6 µg/mL.However, this increase was only observed when gap damage was included and lies within the historical control range. There was no dose-response relationship and this increase was not seen in the first test or at the later sampling time. In addition, the mean frequency of aberrant cells in the solvent controls in this test was slightly low when compared with the average historical control values. Therefore it is concluded that this increase is not treatment related. All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells. It is concluded that Salicynalva has shown no evidence of clastogenic activity in this in vitro cytogenetic test.

Mouse Lymphoma assay: The genotoxicity in mammalian cells was investigated using the mouse lymphoma assay was conducted according to OECD 476 guideline. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range. The positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. In the absence of S9-mix, Salicynalva did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. In the presence of S9-mix, Salicynalva did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation. Therefore Salicynalva is considered to be non-genotoxic in this test.


Justification for selection of genetic toxicity endpoint
The Ames test is one of the three genotoxicity assays needed to assess this endpoint.

Justification for classification or non-classification

Based on the negative results in the assays assessing genotoxicity in bacteria and in mammalian cells and the negative results for chromosomal aberration Salicynalva does not need to be classified for genotoxicity when considering the criteria outlined in Annex I of 1272/2008/EC (CLP) and Annex VI of 67/548/EEC (DSD).