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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
from November 9, 2001 to March 07, 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was performed according to test guideline in compliance with GLP with FAT 41030 a structural analogue of FAT 41045. Both substances are very similar in their chemical structure and, as demonstrated, in a number of physicochemical properties. Therefore, this study is used for read-across thus avoiding duplicate tests.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
FAT 41'030/A

Method

Target gene:
Salmonella typhimurium histidine (his) and the E. coli tryptophan (trp)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver microsome fraction
Test concentrations with justification for top dose:
33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
for all strains with metabolic activation
Positive control substance:
other: 2-aminoanthracene, 2-AA
Remarks:
2.5 µg/plate, 10 µg/plate in strain WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
4 µl/plate for WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
without s9
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPO
Remarks:
10µg/plate in TA 98, 50 µg/plate in TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
without s9
Positive control substance:
sodium azide
Remarks:
10 µg/plate for TA1535, TA 100
Details on test system and experimental conditions:
The bacterial strains TA 1535, TA 1537, and TA 100 were obtained from Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 98 was obtained from E. Merck (D-64293 Darmstadt). The Escherichia coli strain WP2 uvrA was obtained from Dr. Heinz Träger, Kno" AG, D-67008 Ludwigshafen.
summarised the mutations of the TA strains and the E. coH strain
Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA 1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA 98 his D 3052; rfa~; uvrB-;R-factor " "
TA 1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA 100 his G 46; rfa-; uvrB-;R-factor " "
Escherichia coli
WP2 uvrA trp-; uvrA-: base-pair substitutions and others
Pre-Experiment for Toxicity
To evaluate the toxicity of the test item apre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each, except, for strain TA 1537 where only 6 concentrations were tested (due to technical reasons). The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The pre-experiment is reported as main experiment I, if the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.
59 Mix
Before the experiment an appropriate quantity of s9 supernatant was thawed and mixed with s9 co-factor solution. The amount of s9 supernatant was 15% v/v in the cultures.
Cofactors are added to the s9 mix to reach the following concentrations in the 89 mix:
8mM MgCI2
33 mM KCI
5 mM Glucose-6-phosphate
5mM NAOP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
Ouring the experiment the s9 mix was stored in an ice bath. The s9 mix preparation was performed according to Ames et al.(5).
Dose Selection
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I since no relevant toxic effects were observed and 5000 µg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades. The following concentrations were tested:
33; 100; 333; 1000; 2500; and 5000 µg/plate
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, BIOSYS GmbH, D-61184 Karben). The counter was connected to an IBM AT compatible PC with printer to print out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates (see tables of results). Due to precipitation and the intense colour of the test item the revertant colonies were counted manually at 100 µg/plate and above.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
only for TA 98
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
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with and without metabolic activation for TA 98

Based on the results given in this study, FAT 41030/A is considered to be mutagenic in this Salmonella typhimurium and Escheria coli reverse mutation assay for salmonella typhimurium strain TA98 but not for strains TA 1535, TA 1537, TA 100 and Escheria coli strain WP2r uvrA.
Executive summary:

This study was performed to investigate the potential of FAT 41030/A to induee gene mutations aeeording to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escheria coli strain WP2 uvrA. An additional experiment was performed with strain TA 98 using the plate incorporation method. The results of this additional experiment are included in this report as experiment III. The assay was performed with and without liver microsomal activation. In strain TA 1537 no bacterial growth was observed in experiment I and an additional experiment was performed. Due to the erratic result obtained in this experiment these data had to be dismissed and a third experiment was performed according to the plate incorporation method. The results of this experiment are included in this report as part of experiment I. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to5000 µg/plate with and without metabolic activation in both independent experiments.No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony was observed following treatment with FAT 41030/A at any dose level, neither in the presenee nor absence of metabolic activation (S9 mix), except, for strain TA 98 where an increase in revertant colony numbers was observed with and without metabolic activation in all experiments.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item induced gene mutations by frameshifts in the genome of the strain TA 98.