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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test: read-across, non mutagenic (OECD 471, GLP, K, rel. 1) up to 5000 µg/plate in S. typhimurium TA 1537, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included as attachment to section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physico-chemical, (eco)toxicological and environmental fate properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance is one of the constituents of the source substance, together with isomers: the target substance is a mono-constituent, individual optical isomer, while the source substance is defined as a multi-constituent, with three pairs of racemate.

3. ANALOGUE APPROACH JUSTIFICATION
 In vitro gene mutation study in bacteria - Ames test
The source substance and the target substance are isomers. Based on structural and physico-chemical similarities, it is considered appropriate to read-across data from the source substance.
No (reverse) gene mutations were detected in the Ames test performed on the source substance.
The study design (OECD 471, GLP) is adequate and reliable for the purpose of the prediction based on read-across. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the result of the Ames test conducted with the source substance is highly likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VIII, 8.4.1.

4. DATA MATRIX
Cf. attachment to section 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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: Test material vapour pressure (0.04 Pa) is too low to expect a significant effect of evaporation on test results. Test material is not classified as volatile according to the criteria of the Directive 1999/13/EC.
- Water solubility: Test material was solubilized in DMSO to improve solubility
- Precipitation: a precipitate (oily in appearance) was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: the test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-).

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and positive controls. The comparison was made with the historical control ranges for 2003 and 2004 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: the test material caused no visible reduction in the growth of the bacterial background lawn at any dose level, although slight decreases in revertant colony frequency were noted to several of the Salmonella strains at the upper dose level

Preliminary toxicity Test:

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

 

The number of revertant colonies for the preliminary toxicity test were:

Metabolic Activation

Strain

Dose (μg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

133

120

141

138

142

136

133

120

107

120P

107P

+

TA100

87

82

98

94

113

87

94

126

89

80P

76P

-

WP2uvrA-

21

18

18

22

15

22

25

20

17

25P

16P

+

WP2uvrA-

29

15

21

24

17

13

18

22

26

22P

27P

Mutation test:

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 (See Graphs & Tables of results in “Attached background material”) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5 (See Graphs & Tables of results in "Attached background material").

The test material caused no visible reduction in the growth of the bacterial background lawn although a slight decrease in the frequency of revertant colonies was noted to the majority of the Salmonella strains at the upper dose levels. The test material was, therefore, tested up to the maximum recommended dose level of 5000ug/plate. A precipitate (oily in appearance) was observed at and above 1500ug/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Conclusions:
The test item is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA-. The same conclusion applies to the target substance.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E.coli strain WP2 uvrA- were exposed the test material diluted in DMSO both in the presence and absence of a metabolic activation system (10% liver S9 in standard co-factors) using the plate incorporation method. The dose range for the range-finding test was determined in a preliminary toxicity assay and was 15 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn although slight decrease in the frequency of revertant colonies was noted to the majority of the Salmonella strains at the upper dose levels. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A precipitate (oily in appearance) was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

 

Under the test condition, the test material is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA-.

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

The same conclusion applies to the target substance.

Read-across justification is attached to Iuclid section 13.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2005-06-1 to 2005-08-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 471 and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP (inspection date: 2002-12-02 / signature date: 2003-02-13)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine gene for Salmonella and tryptophan gene for E.coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from Sprague-Dawley rats treated with phenobarbitone/-naphtoflavone
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
Range-finding test: 50, 150, 500, 1500 and 5000 μg/plate.
Main test: 50, 150, 500, 1500 and 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test substance immiscible in water. Well known solvent/vehicle not reacting with the test substance.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
See table 7.6.1/1
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
See table 7.6.1/1
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: ca. 48 hours at 37°C

NUMBER OF REPLICATIONS: triplicate plates per dose level

DETERMINATION OF CYTOTOXICITY
- Method: growth assessment of the bacterial background lawn

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

OTHER: ACCEPTANCE CRITERIA: The reverse mutation assay was considered valid if the following criteria were met:
1. All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (according to historical control 2003 & 2004).
2. The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
3. All tester strain cultures should be in the approximate range of 1 to 9.9 billion bacteria per ml.
4. Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
5. There should be a minimum of four non-toxic test material dose levels.
6. There should be no evidence of excessive contamination.
Evaluation criteria:
Dose-related increase in revertant frequency over the dose range tested and/or reproducible at one or more concentrations in at least one bacterial strain with or without metabolic activation.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
none
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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: Test material vapour pressure (0.04 Pa) is too low to expect a significant effect of evaporation on test results. Test material is not classified as volatile according to the criteria of the Directive 1999/13/EC.
- Water solubility: Test material was solubilized in DMSO to improve solubility
- Precipitation: a precipitate (oily in appearance) was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: the test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-).

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and positive controls. The comparison was made with the historical control ranges for 2003 and 2004 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: the test material caused no visible reduction in the growth of the bacterial background lawn at any dose level, although slight decreases in revertant colony frequency were noted to several of the Salmonella strains at the upper dose level

Preliminary toxicity Test:

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

 

The number of revertant colonies for the preliminary toxicity test were:

Metabolic Activation

Strain

Dose (μg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

133

120

141

138

142

136

133

120

107

120P

107P

+

TA100

87

82

98

94

113

87

94

126

89

80P

76P

-

WP2uvrA-

21

18

18

22

15

22

25

20

17

25P

16P

+

WP2uvrA-

29

15

21

24

17

13

18

22

26

22P

27P

Mutation test:

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 (See Graphs & Tables of results in “Attached background material”) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5 (See Graphs & Tables of results in "Attached background material").

The test material caused no visible reduction in the growth of the bacterial background lawn although a slight decrease in the frequency of revertant colonies was noted to the majority of the Salmonella strains at the upper dose levels. The test material was, therefore, tested up to the maximum recommended dose level of 5000ug/plate. A precipitate (oily in appearance) was observed at and above 1500ug/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Conclusions:
The test item is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA-.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E.coli strain WP2 uvrA- were exposed the test material diluted in DMSO both in the presence and absence of a metabolic activation system (10% liver S9 in standard co-factors) using the plate incorporation method. The dose range for the range-finding test was determined in a preliminary toxicity assay and was 15 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn although slight decrease in the frequency of revertant colonies was noted to the majority of the Salmonella strains at the upper dose levels. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A precipitate (oily in appearance) was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

 

Under the test condition, the test material is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA-.

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

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

Additional information

Table 7.6/1: Summary of genotoxicity test on the source substance

Test n°

Test / Guideline

Reliability

Focus

Strains tested

Metabolic activation

Test concentration

Statement

1

 

Safepharm, 2005

Ames Test

(OECD 471)

K, rel. 1

Gene mutation

TA 1535,

TA 1537,

TA 98,

TA 100

E. coli WP2uvrA

-S9

+S9

Up to limit concentration

-S9 : non mutagenic

+S9 : non mutagenic

Gene mutation Assays (Tests n° 1):

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 on the source substance (Test n°1, see Table 7.6/1). 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 source substance, either in the presence or absence of metabolic activation. The source substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The source substance, and by analogy the target substance are therefore considered as non-mutagenic according to the Ames test.

(see Iuclid section 13 for read-across justification).

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP).

Self-classification:

Based on the available information on the source substance, no additional classification is proposed regarding germ cell mutagenicity according to the CLP and to the GHS.