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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to cytotoxic concentrations in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.

- Ames Test (similar to OECD 471, rel.2): non mutagenic up to the maximum concentration in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 April to 5 May, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well conducted and well described study in accordance with GLP and OECD Guideline 471 without any deviation.
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)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
11 March 2005
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium histidine (his) reversion system and the Escherichia coli tryptophan (trp) reversion system
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):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10 % v/v S9; S9 fraction was prepared from liver homogenates of Sprague Dawley rat induced with Aroclor 1254
Test concentrations with justification for top dose:
Assay No. 1 (plate incorporation method): 30, 90, 300, 900 and 3000 μg/plate, with or without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (uvr A-)Assay No. 2 (pre-incubation method): 30, 90, 300, 900 and 3000 μg/plate, with or without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (uvr A-)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol - Test substance preparation:For the first bacteriostatic activity control, the highest dose studied is 5000 μg/plate. A solution at 200 mg/mL is prepared with ethanol.For the two other bacteriostatic activity controls and mutagenic assays, the highest dose studied is 3000 μg/plate. A solution at 120 mg/mL is prepared with ethanol.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: cis-Platinum (II) Diammine Dichloride
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other:
Remarks:
with metabolic activation
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: Strains of Salmonella typhimurium and Escherichia coli are obtained from "Unité de Programmation Moléculaire et Toxicologie Génétique" (CNRS UA 144) (Institut Pasteur).METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubationDURATION- Preincubation period: Pre-incubation assay where the test substance was preincubated with the test strain, and 500 μL of S9-mix fraction for 1 hour at 37° C prior to mixing with the overlay agar and pouring onto the surface of the minimal agar plate.- Exposure duration: Plates were incubated at 37 °C over a 48 hour periodNUMBER OF REPLICATIONS: 3 plates/dose for all groupsDETERMINATION OF CYTOTOXICITY- Method: reduced number of spontaneous reversions indicating the bacteriostatic activity.- OTHER:- Checking strains: The genotype of bacterial strains was checked for Histidine and tryptophan requirements; Loss of cell wall LPS (rfa mutation) measuring crystal violet inhibition for Salmonella typhimurium strains; Ampicillin resistance for the strains which have the pKM 101 plasmide; Δuvr B mutation i.e. U.V.B sensitivity for Salmonella typhimurium and Δuvr A mutation i.e. U.V.A sensitivity for Escherichia coli; Spontaneous revertant rate- After a 48 hour incubation period at 37° C, revertant colonies per plate are counted (n = 3).- Sterility tests: The highest concentration and dilutions of the test substance (100 μL) were tested for sterility.- Data were presented as the number of revertant colonies (mean ± standard deviation) per plate. The following ratio was calculated: R = Number of revertant colonies in the presence of the product / Number of revertant colonies in the absence of the product.
Evaluation criteria:
The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101) strains with and/or without metabolic activation.The validity criteria are as follows :- bacteriostatic activity of the highest concentration shall be equal to or less than 75 %, - the spontaneous reversion rate of the absolute negative control shall comply with the laboratory’s historical control data.- the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with laboratory’s historical control data.The result of the test is considered positive if a concentration – related increase is obtained in one, or several of the 5 strains, with and/or without metabolic activation; a mutagenic effect is taken into account for a given concentration of the test substance if the number of revertant colonies is at least two fold that of spontaneous revertant colonies number for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101), and three fold for TA 1535 and TA 1537.All results must be confirmed in an independent experiment.
Statistics:
None
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
BACTERIOSTATIC ACTIVITY CONTROL (STRAIN TA 100)Bacteriostatic activity control (strain TA 100): In the first control, an important bacteriostatic activity of 94 % is observed in the presence of the test substance at 5000 μg/plate. The test substance is used at the following doses 3000, 900, 300, 90 and 30 μg/plate for bacteriostatic activity control 2 and 3. Then a bacteriostatic activity of 72 % compatible with the maximum level acceptable 75 % is observed, in the presence of the test substance at 3000 μg/plate.The test substance is tested at the following doses: 3000, 900, 300, 90 and 30 μg/plate.HISTORICAL CONTROL DATA - See historical control table in "Attached background material" sectionMUTAGENICITY RESULTS- There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental historic values obtained in the laboratory. There is no evidence of any increase in the number of revertant colonies in the presence of the test substance (3000, 900, 300, 90 and 30 μg) without and with metabolic activation for bacterial strains in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101).- Results were confirmed in a second independent experiment.ADDITIONAL INFORMATION ON CYTOTOXICITY:- In the presence of the test substance at 3000 μg/plate we observe for some bacterial strains like Salmonella typhimurium TA 1535, TA 98 and Escherichia coli WP2(uvrA-) (pKM 101) a significant decrease of the number of spontaneous reversions without or with metabolic activation which confirmed the bacteriostatic activity observed at this dose.OTHERS:Sterility test did not show any bacterial growth in presence of all substance doses / S9 mix.Genotoxicity testing: see tables 3.1 to 3.4 for TA 1535; 4.1 to 4.4 for TA 1537; 5.1 to 5.4 for TA 98; 6.1 to 6.4 for TA 100; 7.1 to 7.4 for Escherichia coli in "Attached background material" section.

None

Conclusions:
Under the test conditions, test substance is not considered as mutagenic with and without metabolic activation in S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2uvrA) strains.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli (WP2uvrA) were exposed to test substance at the following concentrations. 

Assay No. 1 (plate incorporation method): 30, 90, 300, 900 and 3000 μg/plate, with or without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (uvr A-)

Assay No. 2 (pre-incubation method): 30, 90, 300, 900 and 3000 μg/plate, with or without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (uvr A-) 

 

Metabolic activation system used in this test was10 % (v/v) S9 mix; S9 fraction prepared from liver homogenates of rats induced with Aroclor 1254. Negative, vehicle and positive control groups were also included in mutagenicity tests.

 

There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental historic values obtained in the laboratory.

There is no evidence of any increase in the number of revertant colonies in the presence of the test substance (3 000, 900, 300, 90 and 30 μg) without and with metabolic activation for bacterial strains in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101).

In the presence of the test substance at 3 000 μg/plate we observe for some bacterial strains like Salmonella typhimurium TA 1535, TA 98 and Escherichia coli WP2(uvrA-) (pKM 101) a significant decrease of the number of spontaneous reversions without or with metabolic activation which confirmed the bacteriostatic activity observed at this dose. Results were confirmed in a second independent experiment.

 

Under the test conditions, test substance is not considered as mutagenic with and without metabolic activation in these bacterial systems.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Gene mutation Assays :

A Key study was identifed (Phycher, 2006). A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 with the registered substance. For each bacteria strain, concentrations of 30, 90, 300, 900 and 3000 µg/plate were used for the plate incorporation method and for the pre-incubation method.

There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental historic values obtained in the laboratory. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test condition, with any dose of the substance, either in the presence or absence of metabolic activation. In the presence of the test substance at 3 000 μg/plate we observe for some bacterial strains like Salmonella typhimurium TA 1535, TA 98 and Escherichia coli WP2(uvrA-) (pKM 101) a significant decrease of the number of spontaneous reversions without or with metabolic activation which confirmed the bacteriostatic activity observed at this dose. Results were confirmed in a second independent experiment.

In a supporting study performed similarly to the OECD guideline No. 471, Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and Escherichia coli (WP2uvrA) strains were exposed to the test substance in a first test at concentrations of 1.2, 4.9, 20, 78, 313, 1250 and 5000 µg/plate with and without metabolic activation. In a second test, all strains were exposed from 156 to 5000 µg/plate with metabolic activation, S. typhimurium TA 1537, TA 98 and E. coli WP2 strains were exposed at the same concentration without metabolic activation and S. typhimurium TA 100 and TA 1535 strains were exposed from 39 to 2500 µg/plate without metabolic activation.

No significant increases in the frequency of revertant colonies were recorded in this study.

CONCLUSION: The substance is considered as non-mutagenic according to the Ames test.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.