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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
The test substance was found to be non mutagenic based on the results of a bacterial reverse mutation test and a mammalian chromosomal aberration test in vitro.
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 April 19, 2006 to July 13, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Prival et al (Prival MJ and Mitchell VD. Analysis of a method for testing azo dyes for mutagenicity in Salmonella typhimurium in the presence of flavine mononucleotide and hamster liver S9. Mutation research, 97: 103-116; 1982)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from rat liver (plate incorporation test; preincubation test) and S9-mix from hamster liver (preincubation test)
Test concentrations with justification for top dose:
Concentration range in the main test with and without metabolic activation: 51.2, 128, 320, 800, 2,000 and 5,000 µg/plate
Vehicle / solvent:
Deionized water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
two solvent control (i.e., DMSO solvent and distilled water) groups were used depending on the solubility of the test substance and the solubility of strain specific positive chemicals
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine (i.e., 4-NPD)
Remarks:
without metabolic activation for strain TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
two solvent control (i.e., DMSO solvent and distilled water) groups were used depending on the solubility of the test substance and the solubility of strain specific positive chemicals
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation for strain TA 100 and TA 1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
two solvent control (i.e., DMSO solvent and distilled water) groups were used depending on the solubility of the test substance and the solubility of strain specific positive chemicals
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation for strain TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
two solvent control (i.e., DMSO solvent and distilled water) groups were used depending on the solubility of the test substance and the solubility of strain specific positive chemicals
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation for strain WP2uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
two solvent control (i.e., DMSO solvent and distilled water) groups were used depending on the solubility of the test substance and the solubility of strain specific positive chemicals
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation (i.e., 10% rat liver) for all strains
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
two solvent control (i.e., DMSO solvent and distilled water) groups were used depending on the solubility of the test substance and the solubility of strain specific positive chemicals
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation (i.e., 30% hamster liver) for strain TA 100, TA 1535, TA 1537 and WP2uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
two solvent control (i.e., DMSO solvent and distilled water) groups were used depending on the solubility of the test substance and the solubility of strain specific positive chemicals
Positive controls:
yes
Positive control substance:
congo red
Remarks:
with metabolic activation (i.e., 30% hamster liver) for strain TA 98
Details on test system and experimental conditions:
ASSAY PROCEDURE

-The main test included an initial mutation test (i.e., plate incorporation test), a confirmatory mutation test (i.e., pre-incubation test using rat liver S9) and an additional confirmatory mutation test (pre-incubation test using hamster liver S9).

-In the main tests the test substance concentrations, including the controls (untreated, solvent and positive reference) were tested in triplicate.

Test substance concentrations and controls in the main tests (i.e., initial, confirmatory and additional confirmatory tests)
-Based on the results of the preliminary tests, 100 mg/mL stock solution was prepared from the test substance with distilled water, which was diluted in five steps. In the main tests six different amounts of the test substance were tested arranged in a geometric series with a factor of 0.4 between test points. The highest concentration of the test substance in this study was 5,000 µg test substance/plate.

Test substance concentrations tested:
Test substance (No. of concentration) Concentrations tested (µg/plate)
1 5,000
2 2,000
3 800
4 320
5 128
6 51.2

Test solutions were freshly prepared at the beginning of the experiments.

Control groups used in the main tests (i.e., initial, confirmatory and additional confirmatory tests)

Strain-specific positive and negative (solvent) controls, both with and without metabolic activation were included in each test. In addition, untreated control was used demonstrating that the chosen solvent induced no deleterious or mutagenic effects.

Experimental Method
1. Procedure for exposition in the initial mutation test

-A standard plate incorporation procedure was performed, as an initial mutation test.
-Bacteria (i.e., cultured in Nutrient broth No.2.) were exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (i.e., 3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test substance and other components were prepared freshly and added to the overlay (45°C).

-The content of the tubes:
Top agar 2,000 µL
Solvent or solution of test substance or positive controls 50 µL
Overnight culture of tester strain 100 µL
Phosphate buffer (pH:7.4) or S9 mix 500 µL


-This solution was mixed up and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activation and an activation test conditions and each of them with the addition of negative and positive controls. After preparation the plates were incubated at 37°C for 48 h.

2. Procedure for exposition in the confirmatory mutation test

-A pre-incubation procedure was performed as a Confirmatory Mutation Test since the result of the Initial Mutation Test was negative.
-In the pre-incubation procedure the test substance, S9, and bacteria are incubated at higher concentrations than in the standard plate incorporation method. Pre-incubation procedures have equal or greater sensitivity than the plate incorporation assays.
-Before the overlaying of the test substance, the bacterial culture and the S9 mix (i.e., containing rat liver S9) or phosphate buffer were added into appropriate tubes to provide direct contact between bacteria and the test substance (in its solvent). These tubes were gently mixed and incubated for 20 min at 37°C in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes, the content was mixed up and poured onto minimal glucose agar plates as described above for the standard plate incorporation method.
-The entire test consisted of non-activation and activation test conditions and each of them with the addition of negative and positive controls. After preparation the plates were incubated at 37°C for 48 h.

3. Procedure for exposition in the additional confirmatory mutation test

-A pre-incubation procedure was performed, as an additional confirmatory mutation test because of the negative results of the initial mutation test.
-Before the overlaying of the test substance, the bacterial culture and the S9 mix (containing hamster liver S9) or phosphate buffer were added into appropriate tubes to provide direct contact between bacteria and the test substance (in its solvent). These tubes were mixed and incubated for 20 min at 30°C in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes, the content mixed and poured onto minimal glucose agar plates as described above for the standard plate incorporation method.
-The entire test consisted of non-activation and activation test conditions and each of them with the addition of negative and positive controls. After preparation the plates were incubated at 37°C for 48 h.
Evaluation criteria:
EVALUATION OF EXPERIMENTAL DATA

The colony numbers on the control, positive control and the test plates were determined. The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test substance and for the controls using Microsoft Excel™ software.

A test substance is considered as mutagenic if:
- a dose-related increase in the number of revertants occur and/or
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain TA 100 the number of reversions is at least twice as high as the reversion rate of the solvent control
- in strain TA98. TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the solvent control

According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results; a statistical evaluation of the results is not regarded as necessary.

A test substance producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the test points is considered non-mutagenic in this system.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
(in plate incorporation test and peincubation test)
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
(in plate incorporation test and peincubation test)
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRELIMINARY STUDY (RANGE FINDING TEST)

-In the preliminary range finding test plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 test strains under activation and non-activation conditions.

-In the range finding study the concentrations examined were: 5,000; 2,500; 1,250; 625; 312.5; 156.25; 78.13; 39.06 and 19.53 µg/plate.
The revertant colony numbers were slightly increased compared to the revertant colony numbers of the solvent control plates in case of S. typhimurium TA98 at only one concentration level, with addition of metabolic activation at 78.131 µg/plate. Slightly decreased revertant colony numbers were noticed in S. typhimurium TA98, with metabolic activation at 2,500 µg/plate, without metabolic activation at 312.5 µg/plate.

-There was no increase or decrease in revertant colony number compared to the solvent control plates al S. typhimurium TA 100.


INITIAL MUTATION TEST
-In the initial mutation assay the plate incorporation method was used. The experiment was carried out using S. typhimurium strains (TA98, TA 100, TA 1535, TA 1537) and Escherichia coli WP2 uvrA strain, in presence and absence of metabolic activation (S9 Mix) using appropriate positive and negative controls.

-The examined concentrations were: 5,000; 2,000; 800; 320; 128 and 51.20 µg/plate.

-Sporadic changes were observed, such as non-significant increases and decreases in revertant colony numbers compared to the solvent control values in all examined bacterium strains with exception of S. typhimurium TA 100.

-These variations were not dose dependant and remained far below the threshold for being positive and should thus be considered as reflecting the spontaneous biological variability.

-The highest increase was observed in E. coli WP2 uvrA at the concentration of 5,000 I µg/plate (-S9), where the mutation factor value was: 1.49.

CONFIRMATORY MUTATION TEST (USING RAT LIVER S9)

-In the confirmatory mutation assay the pre-incubation method was used. The experiment was carried out using Salmonella typhimurium strains (TA98, TA 100. TA1535. TA 1537) and Escherichia coli WP2 uvrA strain. in presence and absence of metabolic activation (S9 Mix containing rat liver S9) using appropriate positive and negative controls.

-The examined concentrations were: 5,000; 2,000; 800; 320; 128 and 51.20 µg/plate.

-The revertant colony numbers were significantly increased without metabolic activation at the highest concentration level, at 5,000 µg/plate in case of strains whose auxotrophy was caused by frameshift mutations (S. typhimurium TA98 and TA1537).

-While the observed increases were dose-related in S. typhimurium TA98 in the concentration range of 5,000-320 µg/plate, in TAI537 the dose-relationship was equivocal. In S. typhimurium TA 1537 increased rates were observed at: (-S9) 5,000. 2,000 and 320 µg/plate. The highest increase was observed in S. typhimurium TA 1537 at the concentration of 5,000 µg/plate, where the mutation factor value was: 2.14.

-The high mutation factor values (mutation rates) remained below the threshold for being positive*.
*The: increase is biological relevant in the case of S. typhimurium TA98 and TAI537 when the number of reversion is at least three times higher as compared to the reversion rate at the solvent control.

-Additionally, sporadic, non-significant increases in revertant colony numbers compared to the solvent control values (the mutation factor values remained in the range of 1.20-1.30) were observed at different concentration levels in S typhimurium TA1535 and E. coli WP2 uvrA. These changes were of minor intensity, not dose related and without any biological significance. There was no increase or decrease in revertant colony number compared to the solvent control plates at S. typhimurium TA100.
Slight decreases in revertant colony numbers could be observed only in S typhimurium TA1537.

ADDITIONAL CONFIRMATORY MUTATION TEST (USING HAMSTER LIVER S9)

-In the additional confirmatory mutation assay the pre-incubation method was used. The experiment was carried out using S typhimurium strains (TA98, TA 100. TA 1535. TA 1537) and Escherichia coli WP2 uvrA strain, in presence and absence of metabolic activation (S9 Mix containing hamster liver S9) using appropriate positive and negative controls.
The examined concentrations were: 5,000; 2,000; 800; 320; 128 and 51.20 µg/plate.

-Increased revertant colony numbers compared to that of the solvent control values were observed without metabolic activation in the case of S. typhimurium TA98 and TA 1537 at the concentration levels of 5,000 and 2,000 µg/plate in E. coli WP2 uvrA at 5,000 µg/plate, with metabolic activation in S typhimurium TA98 at 2,000 µg/plate, and in TA 1535 at 320 µg/plate.

-The revertant colony numbers were significantly increased at 5,000 and 2,000 µg/plate (-S9) in the case of S. typhimurium TA 1537. The mutation factor values were 2.08 and 1.62. These high mutation factor values remained below the threshold for being positive. The further increases were evaluated as sporadic non-significant increases.

-There was no increase or decrease in revertant colony number compared to the solvent control plates at S. typhimurium TA 100.

-The observed decreases in revertant colony numbers compared to that of the solvent control values in S. typhimurium TA1535 and TA1537 with addition of metabolic activation at 5,000 and 2,000 µg/plate could not be evaluated unequivocally as slight inhibitory effect.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Results of the solubility test.

 

Concentration of test substance in distilled water (mg/mL)

Solubility in distilled water

Solubility in the top solution (test solution 50 µL + phosphate buffer 500 µL + top agar 2 mL

Test substance concentration in the test tube: µg/tube

100

Solution

Solution

 5,000

Conclusions:
Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay.
Executive summary:

An in vitro study was performed to investigate the potential of the test substance to induce gene mutations according to OECD Guideline 471, EU Method B.13/14, EPA OPPTS 870.5100 and Prival et al, in compliance with GLP.

 

Three mutagenicity studies were conducted, one as the plate incorporation method and the other two as a preincubation test. The studies were performed in the absence and presence of a metabolizing system derived from rat or hamster liver homogenate. The substance was tested for mutagenic effects without and with metabolic activation at six concentrations in the range of 51.2 - 5,000 µg/plate in all assays.

 

In the plate incorporation test, the test substance did not result in relevant increases in the number of revertants in any of the bacterial strains in the absence and presence of the metabolic activation (rat liver S9-mix (10% (v/v)). Also, in the preincubation tests no relevant increase in the number of revertants was observed in any of the bacterial strains in the absence and presence of the metabolic activation (rat liver S9-mix (10% (v/v and/or hamster liver S9 -mix (30% (v/v)). Further, the spontaneous revertant colony numbers of solvent control plates without S9 mix were within the historical control data range. The reference mutagens showed the expected increase in induced revertant colonies

 

Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay.

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

Additional information

Additional information from genetic toxicity in vitro:

An in vitro study was performed to investigate the potential of the test substance to induce gene mutations according to OECD Guideline 471, EU Method B.13/14, EPA OPPTS 870.5100 and Prival et al, in compliance with GLP. Three mutagenicity studies were conducted, one as the plate incorporation method and the other two as a preincubation test. The studies were performed in the absence and presence of a metabolizing system derived from rat or hamster liver homogenate. The substance was tested for mutagenic effects without and with metabolic activation at six concentrations in the range of 51.2 - 5,000 µg/plate in all assays. In the plate incorporation test, the test substance did not result in relevant increases in the number of revertants in any of the bacterial strains in the absence and presence of the metabolic activation (rat liver S9-mix (10% (v/v)). Also, in the preincubation tests no relevant increase in the number of revertants was observed in any of the bacterial strains in the absence and presence of the metabolic activation (rat liver S9-mix (10% (v/v and/or hamster liver S9 -mix (30% (v/v)). Further, the spontaneous revertant colony numbers of solvent control plates without S9 mix were within the historical control data range. The reference mutagens showed the expected increase in induced revertant colonies. Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay (Vertesi, 2006).

A study was conducted to investigate the potential of the test substance to induce chromosome aberrations in Chinese hamster ovary cells (i.e., CHO-KI cells) according to OECD Guideline 473 in compliance with GLP. Two independent assays were conducted, one with treatment/sampling times of 3/20 h and other one with treatment/sampling times of 20/28 h and 3/28 h. The test substance was dissolved in Ham's F12 medium and tested at the concentrations ranging from 78.12-2,500 µg/mL with and without metabolic activation, based on the results of preliminary testing for solubility and toxicity. Positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay and the efficacy of the S9-mix. The test substance did not induce an increase in the number of chromosomal aberrations following 3 h treatment time either in the absence or in the presence of metabolic activation up to and including cytotoxic concentrations. Also, no chromosome aberrations were observed without S9 mix up to the highest tested concentration of 312.50 µg/mL, following a long treatment period of 20 h. Moreover, there was no increase in the rate of polyploid or endoreduplicated metaphases in either assay in the presence or absence of metabolic activation. Under the study conditions, the test substance did not induce structural chromosome aberrations in Chinese hamster ovary cells (Beres, 2006).


Justification for selection of genetic toxicity endpoint
Guideline-compliant study conducted according to GLP.

Justification for classification or non-classification

Based on the results of a bacterial reverse mutation test and a mammalian chromosomal aberration test, the substance does not need to be classified for mutagenicity according to the EU CLP criteria (EC 1272/2008) as well as EU Directive 67/548/EEC.