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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:
experimental study
Adequacy of study:
key study
Study period:
July 15 to July 21, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline test with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification: Epikote P
Structure : Substance is reaction product of Epikote 828 (CAS 25068-38-6)
n= 0 (90 mol%): n= 1 (10 mol%), and biphenyl-4-ol (CAS 92-69-3)
Molecular formula : C45H44O6 (ca. 52.5% by weight) Lower molecular weight approximately 7.5%; higher molecular weight approximately 40% by weight
Molecular weight: 680.8 (main component); Approximately 900 (number average resin)
CAS Number : 161308-15-2 (main component)
Description : Pale yellow powder
Batch : 8401500-710036 (taken from label)
Purity: > 99.8%
Test substance storage: At room temperature in the dark
Stability under storage conditions :stable
Expiry date : 01 December 2010













Method

Target gene:
Salmonella typhimurium bacterial strains resulting in histidine-independent strains, and in a gene of tryptophan-requiring Escherichia coli bacterial
strain resulting in a tryptophan-independent strain.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomai enzymes
Test concentrations with justification for top dose:
10, 33,100, 333,1000, 3330 and 5000 µg/plate
Vehicle / solvent:
dimethyl sulfoxide (SeccoSolv, Merck, Darmstadt, Germany).
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide
True negative controls:
no
Positive controls:
yes
Remarks:
-s9-mix
Positive control substance:
sodium azide
Remarks:
5 µg for TA 1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
-s9-mix
Positive control substance:
9-aminoacridine
Remarks:
60µg for TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
-s9-mix
Positive control substance:
2-nitrofluorene
Remarks:
10µg
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
-s9-mix
Positive control substance:
monomeric acrylamide
methylmethanesulfonate
Remarks:
650µg for TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
-s9-mix
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
10 µg for WP2uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
+s9-mix
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
2.5 µg for TA 1537(5% s9-mix), TA 1535(5% and 10% S9); 2.5 µg for TA 100 (10% s9-mix); 1 µg for TA 98(5% s9-mix) and TA 100(5% s9-mix); 10 µg for WP2uvrA(5 and 10% s9-mix); 5 µg for TA1537(10% s9-mix)
Details on test system and experimental conditions:
Test system: Salmonella typhimurium bacteria and Escherichia coli bacteria
Rationale: Recommended test system in international guidelines (e.g. OECD, EEC and MITI).
Source:
Salmonella typhimurium strains:
Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames):
TA100 received on 14-06-2006, used batch: TA100.130208
TA98 received on 14-06-2006, used batch: TA98.290108
TA1537 received on 14-06-2006, used batch: TA1537. 210807
TA1535 received on 14-06-2006, used batch: TA1535.210807
Escherichia coli strain:
(Master culture from The National Collections of Industrial and Marine
Bacteria, Aberdeen, UK)
WP2uvrA received on 06-02-2008, used batch: EC.270208
The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor= plasmid pKM101 (increases error-prone DNA repair)
The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
The Escherlchia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment (Ref.1). The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.
Stock cultures of the five strains were stored in liquid nitrogen (-196°C).
Metabolic activation system:
Rat liver microsomai enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River, Sulzfeld, Germany.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negat ive response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
Statistics:
no data

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:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
1. First mutation experiment
Epikote P was tested in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA with concentrations of 10, 33,100, 333, 1000, 3330 and 5000 ug/plate in the absence and presence of 5% (v/v) S9-mix.
Precipitate
Precipitation of Epikote P on the plates was observed at the start and at the end of the incubation period at concentrations of 333 ug/plate and upwards.
Toxicity
To determine the toxicity of Epikote P, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Mutaqenicitv
No increase in the number of revertants was observed upon treatment with Epikote P under all conditions tested.
2. Second mutation assay
To obtain more information about the possible mutagenicity of Epikote P, a second mutation experiment was performed in the absence and presence of 10% (v/v) S9-mix. Based on the results of the first mutation experiment, Epikote P was tested up to concentrations of 333 ug/plate.
Precipitate
Precipitation of Epikote P on the plates was observed at the start and at the end of the incubation period at the concentration of 333 ug/plate.
Toxicity
The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
Mutaqenicitv
No increase in the number of revertants was observed upon treatment with Epikote P under all conditions tested.





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):
negative with and without metabolic activation

Based on the results of this study it is concluded that test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

This study was conducted to evaluate the test substance for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidine-independent strains, and in a gene of tryptophan-requiring Escherichia coli bacterial strain resulting in a tryptophan-independent strain. This study was performed based on the OECD guideline of 471 under the GLP conditions. In first mutation assay, test substance was tested at d up to concentrations of 5000 ug/plate in the absence and presence of 5% (v/v) S9-mix. Epikote P precipitated on the plates at dose levels of 333 ug/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the second mutation assay, test article was tested up to concentrations of 333 ug/plate in the absence and presence of 10% (v/v) S9-mix. Test substance precipitated on the plates at the top dose of 333 ug/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Test substance did not induce a significant dose-related increase in the number of revertant (His+ ) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+ ) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

Therefore, it is concluded that Epikote P is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.