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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
GLP study according to OECD Guideline 471 and 472 and EU Methods B.13/14. Ames Experimental Study #1: Positive mutagenicity without S9 activitation for S. typhimurium strains TA1537 and TA98; Positive mutagenicity with S9 activitation for S. typhimurium strains TA98 and TA100; Negative mutagenicity for S. typhimurium strains TA1535, TA100 and E. coli WP2uvrA; and negative mutagenicity for S. typhimurium strains TA1535, TA1537, and E. Coli WP2uvrA. Ames Experimental Study #2: Positive mutagenicity without S9 activitation for S. typhimurium strains TA98; Negative mutagenicity without S9 activitation for S. typhimurium strains TA100, TA1535, and TA1537 and E. coli WP2uvrA; Positive mutagenicity with S9 activitation with S. typhimurium strains TA98 and TA100; Negative mutagenicity with S9 activitation for S. typhimurium strains TA1535, and TA1537 and E. coli WP2uvrA.
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:
13 October 1997 - 23 October 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guidelines 471, 472 and EU Method B.13/14 without deviations and GLP practices.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine-requiring S. typhimurium, and
Tryptophan-requiring E. coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: Agar Plates (0.9 cm) contained 25 mL glucose agar medium. During the test period the strains also contained 12.5 ug/plate biotin and 15 ug/plate histidine. The top agar was composed of Vogel-Bonner Medium E containing 0.6% (w/v) purified agar was heated to dissolve the agar. Samples of 3 mL top agar were transferred into 10 mL glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 deg C.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media: Agar Plates (0.9 cm) contained 25 mL glucose agar medium. During the test period the strains also contained 15 ug/plate tryptophan. The top agar was composed of Vogel-Bonner Medium E containing 0.6% (w/v) purified agar was heated to dissolve the agar. Samples of 3 mL top agar were transferred into 10 mL glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 deg C.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-fraction
Test concentrations with justification for top dose:
Without activation: 100, 333, 1000, 1800, 3330 ug/plate
With activation: 100, 333, 1000, 1800, 3330 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Saline and DMSO
- Justification for choice of solvent/vehicle: No data
Untreated negative controls:
yes
Remarks:
The vehicle of the test substance, being ethanol absolute.
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
methylmethanesulfonate
other: daunomycine and 2-aminoanthracene
Remarks:
See table below for a breakout of the positive control substances used for each strain
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

SELECTION AGENT (mutation assays): Incubation time

NUMBER OF CELLS EVALUATED: colony counting: The revertant coloniew (histidine independent c.q. tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - number of reverants
Evaluation criteria:
The test substance is considered negative (not mutagenic) in the test if:
- The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
- The negative response should be reproducible in at least one independently repreated experiment.

The test substances is considered positive (mutagenic) in the test if:
- It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
- The positive response should be reproducible in at leaset one independently repreated experiment.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
TA1537 without activation had a 2.4-fold increase in the number of revertant (His+) colonies, first exp only. TA98 induced a 2.0 fold, dose-related, increase in both experiments. TA1535, TA100 did not induce dose related increase, both exp.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
TA98 induced a 4.1 fold dose-related increases in the number of revertant (His+) colonies in Exp. 1. Strain TA100 induced a 3.0 fold dose-related increase in Exp. 1. Strain TA1535, and TA1537, did not induce a dose-related increase, both exp.
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:
without
Genotoxicity:
negative
Remarks:
WP2uvrA did not induce a dose-related increase in the number of revertent colonies, in both exp.
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
Genotoxicity:
negative
Remarks:
WP2uvrA, did not induce a dose-related increase, both exp.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance did not precipitate in the top agar. Preceiptation of thes test substace on the plates weas not observed at the start or at the end of the incubation period in tester strain TA100 and WP2uvrA.

RANGE-FINDING/SCREENING STUDIES: (Experiment 1): The test substance was tested in the tester strains TA100 and WP2uvrA wtih concentration of 3, 10, 33, 100, 333, 1000, 3330, and 5000 µg/plate in the absence and presence of S9-mix.

(Experiment 2): Based on the toxicity data of the first range finding study and the first experiment, the following dose range finding was tested in the second experiment. Strains TA1535 and TA100 with and without activation were tested at concentrations of 100, 333, 1000, 1800, 2400 ug/plate. Strain TA1537 with activation were tested at concentrations of 100, 333, 1000, 1800, 3330 ug/plate and without activation were tested at concentrations of 33, 100, 333, 1000, 1300 ug/plate. Strain TA98 with and without activation were tested at concentrations of 100, 333, 1000, 1800, 3330 ug/plate. Based on the toxicity data of the first experiment, the following dose range was tested in the second experiement with the E. coli strain WP2uvrA with and without activation were tested at concentrations of 100, 333, 1000, 3330, and 5000 ug/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: All other concentrations, not present in the table below, and strain Wp2uvrA showed no reduction in the bacterial background lawn or showed no reduction in the number of revertants, which was less than the minimal value of the historical control data range.

The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Experiment 1 Test Results:

Strain

Dose ug/plate

Without activation

With Activation

Bacterial background lawn

Revertant colonies

Bacterial background lawn

Revertant colonies

TA1535

1800

Slight

-1

Slight

-1

3330

Extreme

Extreme 2

Extreme

Extreme 2

TA1537

1000

Slight

-1

-0

-1

1800

Extreme

Extreme 2

Slight

-1

3330

Absent

Complete

Extreme

Extreme 2

TA98

3330

Moderate

Extreme

Moderate

-2

-0= No reduction in the bacterial background lawn.

-1= no reduction in the number of revertant colonies, which was less than the minimal value of the historical control data range.

-2= Microcolonies

-3= Reduction in the number of revertant colonies, compared to the concentration of 1800 ug/plate.

 

Note: All other concentrations, not mentioned here, showed no reduction in the bacterial background lawn or in the number of revertant colonies, which was less than the minimal values of the historical control data range.

Conclusions:
Interpretation of results (migrated information):
positive without metabolic activation S. typhimurium, strains TA1537 and TA98
positive with metabolic activation S. typhimurium, strains TA98 and TA100
negative without metabolic activation S. typhimurium, strains TA1535 and TA100; E. coli, strain WP2uvrA
negative with metabolic activation S. typhimurium, strains TA1535 and TA1537; E. coli, strain WP2uvrA

Based on the results of this study it was concluded that Epyrrol was mutagenic in the Salmonella typhimurium reverse mutation assay and Epyrrol was not mutagic in the Escherichia coli reverse mutation assay.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vitro:

Justification for selection of genetic toxicity endpoint
Based on the results of this study it was concluded that Epyrrol was mutagenic in the Salmonella typhimurium reverse mutation assay and Epyrrol was not mutagic in the Escherichia coli reverse mutation assay.

Justification for classification or non-classification