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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test (OECD 471, GLP, K, rel. 2): non mutagenic up to cytotoxic concentrations in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E. coli WP2(pKM101) and WP2uvrA(pKM101)

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 August 11 to September 25, 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted according to OECD test guideline No. 471 and No. 472 with deviation: in the –S9 tests, toxicity was not achieved at the highest concentration. However, in the preliminary test, at 500 µg/plate there were some revertant colonies for 5 out of 6 strains, but in no case there was an increase in revertant count. Therefore, the result is expected to be the same even if a toxic dose level is achieved. A re-test is not required.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
in the –S9 tests, toxicity was not achieved at the highest concentration
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
yes
Remarks:
in the –S9 tests, toxicity was not achieved at the highest concentration
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
in the –S9 tests, toxicity was not achieved at the highest concentration
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Stored at 4 °C, protected from light and moisture
Target gene:
Histidine and tryptophan
Species / strain / cell type:
other: S. typhimurium TA1535, TA1537, TA98 and TA100; E. coli WP2(pKM101) and WP2uvrA(pKM101)
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 % S9; S9-mix from the livers of male Sprague Dawley OFA rats treated with Aroclor 1254 at 500 mg/kg bw.
Test concentrations with justification for top dose:
- Preliminary toxicity test: 0, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix using direct plate incorporation method.
- Mutagenicity test (direct plate incorporation method): 0, 1.5, 5, 15, 50 and 150 µg/plate in all strains without S9-mix; 0, 5, 15, 50, 150 and 500 µg/plate in all strains with S9-mix.
- Complementary assay: 0, 5, 50, 150 and 500 µg/plate in TA1537 strain with S9-mix using direct plate incorporation method.
- Mutagenicity test (repeat): 0, 1.5, 5, 15, 50 and 150 µg/plate in all strains without S9-mix using direct plate incorporation method and with S9-mix using pre-incubation method.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test material was not soluble in aqueous medium, therefore it was dissolved in DMSO at a concentration of 50 mg/mL. This solution was used at 100 µl/plate giving a final concentration of 5000 µg/plate.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: potassium dichromate
Remarks:
Without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-anthramine
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation

DURATION
- Exposure duration: Plates were incubated for approximately 48 h at 37 °C.

NUMBER OF REPLICATIONS: Single plate/dose for preliminary toxicity study; triplicate plates per dose level for mutation study.

DETERMINATION OF CYTOTOXICITY
- Method: Microscopic examination of the bacterial background lawn.

OTHER EXAMINATIONS:
- Other: Observations of precipitate of the test substance.

OTHER: READING OF RESULTS:
- After 48 h of incubation at 37 °C, the prototrophic mutant colonies that have developed in the plates are counted using a colony counter.
- The results are expressed as the mean number of revertants per plate and, for each concentration of the test product, the following ratio is established: Mean number of revertants per plate in the presence of the test compound / Mean number of revertants per plate in the solvent control.

OTHER: ACCEPTANCE CRITERIA: The reverse mutation assay was considered valid if the following criteria were met:
- The test compound must be sterile: At the highest dose, after spreading out under the conditions of the toxic activity test and after 48 h incubation at 37 °C, no colony must be visible.
- The mean frequency of spontaneous revertants (solvent control) in each strain must be included between the maximum and minimum historical control values.
- The mean frequency of revertants, for each strain, induced by the reference products both with and without metabolic activation must be greater than the lowest limit historical values.
Rationale for test conditions:
In the –S9 tests, toxicity was not achieved at the highest concentration. However, in the preliminary test, at 500 µg/plate there were some revertant colonies for 5 out of 6 strains, but in no case there was an increase in revertant count. Therefore, the result is expected to be the same even if a toxic dose level is achieved. A re-test is not required.
Evaluation criteria:
- A compound causing a positive response proportional to the dose for at least 3 concentrations with, for the highest increase, a value greater than or equal to 2 times (for strains TA 98, TA 100, WP2(pKM101) and WP2uvrA (pKM101)) or 3 times (for strains TA 1535 and TA 1537) the value for the solvent control, is considered positive in the assay.
- Results were analysed by Dunnett's method when mutant number declined markedly at one or two high doses.
- If a substance produced a positive response during a single assay and that result cannot be reproduced in at least 2 independent assays, the initial positive result may be considered as not significant.
Statistics:
Results were analysed by Dunnett's method when mutant number declined markedly at one or two high doses.
Key result
Species / strain:
other: S. typhimurium TA1535, TA1537, TA98 and TA100; E. coli WP2(pKM101) and WP2uvrA(pKM101)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: Dissolved in DMSO
- Precipitation: None
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: The test item presented withtou metabolic activation a strong toxicity demonstrated by an absence of bacterial growth or by an important decrease in the number of revertants at the three highest doses of 5000, 1500 and 500 µg/plate in the 6 strains tested. Under these conditions, the immediately inferior dose of 150 µg/plate which presented no toxicity, was retained as the high dose for the mutagenicity assay without S9-mix in all bacterial strains.
With S9-mix, at two highest doses of 5000 and 1500 µg/plate, a strong toxicity was observed. The inferior dose of 500 µg/plate which presented a moderate toxicity, was retained as the maximum dose for the mutagenicity assay with S9-mix in all bacterial strains.
However, in the first assay with S9-mix without pre-incubation, a strong or moderate toxicity was noted at the dose of 500 µg/plate in all the strains tested. Consequently, in the second assay, the top dose was reduced to 150 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: The frequency of spontaneous revertants (solvent controls) in each strain was within the limits of historical controls.

None

Conclusions:
Under the test condition, the test material is not mutagenic with and without metabolic activation to S. typhimurium strains TA1535, TA1537, TA98,TA100 and E. coli strains WP2(pKM101) and WP2uvrA(pKM101) according to the criteria of the Annex VI of the of the Regulation (EC) No.1272/2008 (CLP) and according to the GHS.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and No. 472 and in compliance with GLP, S. typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli strains WP2(pKM101) and WP2uvrA(pKM101) were exposed to the test material diluted in DMSO both in the presence and absence of metabolic activation system using the plate incorporation method. The dose range was determined in a preliminary toxicity assay and ranged between 1.5-150 µg/plate (without S9-mix) and 5- 500 µg/plate (with S9-mix). In a complementary assay with S9-mix in TA1537 strain, dose range was between 5-500 µg/plate. Mutagenicity test was repeated using dose range between 1.5-150 µg/plate in all strains without S9-mix using direct plate incorporation method and with S9-mix using pre-incubation method.

Vehicle (DMSO) and positive control groups were also included in mutagenicity tests.

A significant increase in the number of revertants was observed in the presence of reference positive substances. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The frequency of spontaneous revertants (solvent controls) in each strain was within the limits of historical controls. Toxic effects were noted at 5000, 1500 and 500 µg/plate (without S9-mix) and at 5000 and 1500 µg/plate (with S9-mix) in all the tested strains.

In the absence of metabolic activation, in two independant assays, no significant increase in the number of revertants was noted in the presence of the test compound.

In the first assay in the presence of metabolic activation without pre-incubation, a slight increase (3 times the number of spontaneous revertants) at the limit of biological significance was noted at 50 µg/plate in TA1537 strain. However, in an independent complementary assay performed under the same operating conditions, no significant increase in the number of revertants was observed in this strain. Therefore, the effect observed in the first assay was not reproducible. In addition, it was not dose-related, not statistically significant and cannot be attributed to a significant mutagenic activity of the test compound. Furthermore, in the same first assay with metabolic activation, no significant increase in the number of revertants was seen in other bacterial strains.

In the second assay in the presence of metabolic activation using pre-incubation method, no significant increase in the number of revertants was observed in all the bacterial strains tested. 

It is noted that in the first assay in the presence of metabolic activation without pre-incubation in strains TA98 and WP2(pKM101) at the highest dose tested of 500 µg/plate and in the second assay with pre-incubation in all the strains tested at the maximum dose or at two first highest doses of 150 and 50 µg/plate, a statistically significant decrease in the number of revertants was found. However, this effect which was due to the toxic activity of the test compound at high doses, had no meaning in terms of mutagenic hazard.

Under the test condition, the test material is not mutagenic with and without metabolic activation to S. typhimurium strains TA1535, TA1537, TA98,TA100 and E. coli strains WP2(pKM101) and WP2uvrA(pKM101) according to the criteria of the Annex VI of the of the Regulation (EC) No.1272/2008 (CLP) and according to the GHS.

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

Table 7.6/1: Summary of genotoxicity tests

Test n°

Test / Guideline

Reliability

Focus

Strains tested

Metabolic activation

Test concentration

Statement

1

 

Institut Pasteur,

1997

Ames Test

(OECD 471)

K, rel. 1

Gene mutation

TA 1535, TA 1537, TA 98,

TA 100,

E. coli WP2 (pKM101), WP2 uvrA (pKM101)

-S9

+S9

Up to cytotoxic concentration

-S9 : non mutagenic

+S9 : non mutagenic

 

Gene mutation Assay (Test n° 1):

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 with the substance (See Table 7.6/1). In the main (–S9) tests, toxicity was not achieved at the highest concentration of 150 µg/plate. However, in the preliminary test, at 500 µg/plate there were some revertant colonies for 5 out of 6 strains (except TA 1537 strain), but in no case there was an increase in revertant count. Therefore, the result was expected to be the same even if a toxic dose level is achieved. A re-test is not required.

In the first assay in the presence of metabolic activation without pre-incubation, a slight increase (3 times the number of spontaneous revertants) at the limit of biological significance was noted at 50 µg/plate in TA1537 strain. However, in an independent complementary assay performed under the same operating conditions, no significant increase in the number of revertants was observed in this strain. Therefore, the effect observed in the first assay was not reproducible. In addition, it was not dose-related, not statistically significant and hence could not be attributed to a significant mutagenic activity of the test compound. Furthermore, in the same first assay with metabolic activation, no significant increase in the number of revertants was seen in other bacterial strains. In the second assay in the presence of metabolic activation using pre-incubation method, no significant increase in the number of revertants was observed in all the bacterial strains tested. In the first assay in the presence of metabolic activation without pre-incubation in strains TA98 and WP2(pKM101) at the highest dose tested of 500 µg/plate and in the second assay with pre-incubation in all the strains tested at the maximum dose or at two first highest doses of 150 and 50 µg/plate, a statistically significant decrease in the number of revertants was found. However, this effect which was due to the toxic activity of the test compound at high doses, had no meaning in terms of mutagenic hazard.

The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic under the conditions ofthe Ames test.

Justification for classification or non-classification

Harmonized classification:

The test material 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 germ cell mutagenicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and according to the GHS.

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