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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:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guidleine Study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial 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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
- Name of test material (as cited in study report): unoxolTM 3,4 Dialdehyde
- Composition of test material, percentage of components: +/- 53% 1,3-cyclohexanedicarboxdialdehyde (CAS 55309-54-1 )
+/- 47% 1,4-cyclohexanedicarboxdialdehyde (CAS 33424-83-8)
- Lot/batch No.: 200502771 -6M
- Expiration date of the lot/batch: 31 December 2006
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions: Stability in ethanol: at least 96 hours
- Storage condition of test material: In refrigerator (2-8°C) in the dark under nitrogen

Method

Target gene:
ultraviolet-repair B gene
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with
Metabolic activation system:
Phenobarbital and B-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 10, 33, 100, 333, 1000 and 3330 µg/plate
Concentration range in the main test (without metabolic activation): 10, 33, 100, 333, 1000 and 2000 µg/plate
Vehicle / solvent:
Solvent: ethanol
Details on test system and experimental conditions:
The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TAI 537 hisC3076 Frameshift
TA98 hisD30521R-factor* Frameshift
TAI 535 hisG46 Base-pair substitutions
TAI 00 hisG461R-factor* Base-pair substitutions
*: R-factor = plasmid pKMlOl (increases error-prone DNA repair)

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)

The Salmonella typhimurium strains were regularly checked to confirm their histidinerequirement,
crystal violet sensitivity, ampicillin resistance (TA98 and TAIOO), UV-sensitivity
and the number of spontaneous revertants.

The Escherichia coli WP,uvrA 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).
Evaluation criteria:
No formal hypothesis testing was done.
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 unless the total number of revertants in tester strain TAl535,
TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative 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 strain TAI 535, TAI 537, 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.
The preceding criteria were not absolt~tea nd other modifying factors might enter into the final
evaluation decision.
Statistics:
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 unless the total number of revertants in tester strain TAl535,
TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative 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 strain TAI 535, TAI 537, 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.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(>= 3330 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(>= 3330 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
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:
Observations:
All bacterial strains showed negative responses over the
entire dose range, i.e. no significant dose-related increase
in the number of revertants in two independently repeated
experiments.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: preliminary test

Any other information on results incl. tables

TABLE 1. EXPERIMENT 1

MEAN NUMBER OF COLONIES/3 REPLICATE PLATES (+/- SD)

Without S-9 MIX

 DOSE (ug/plate)  TA1535  TA1537  TA98  TA100  WP2uvR
 Positive Control  1046 +/- 80  511 +/- 79  859 +/- 269  1225 +/- 60  778 +/- 7
 Solvent Control  7 +/- 2  4 +/- 2  16 +/- 4  116 +/- 5  16 +/- 1
 3        136 +/- 14  23 +/- 6
 10        131 +/- 9  20 +/- 6
 33  9 +/- 5  4 +/- 2  18 +/- 4  125 +/- 3  13 +/- 1
 100  8 +/- 1  4 +/- 1  17 +/- 2  127 +/- 12  21 +/- 3
 333  8 +/- 3  4 +/- 2  18 +/- 4  134 +/- 11  22 +/- 3
 1000  3 +/- 2  7 +/- 6  10 +/- 1  172 +/- 15  18 +/- 3
 2000  0 +/- 0a  0 +/- 0a  0 +/- 0a    
 3330  0 +/- 0a  0 +/- 0a  0 +/- 0a 0 +/- 0a   0 +/- 0a
 5000        0 +/- 0a  0 +/- 0a
                WITH S-9 MIX 1
 Positive Control  148 +/- 12  320 +/- 51  1051 +/- 7  969 +/- 97  278 +/- 20
 Solvent Control  7 +/- 3  5 +/- 2  23 +/- 5  119 +/- 2  21 +/- 3
 3        120 +/- 13  23 +/- 2
 10        129 +/- 9  19 +/- 5
 33  9 +/- 1  3 +/- 1  26 +/- 3 133 +/-14  17 +/- 1
 100  9 +/- 2  3 +/- 1  26 +/- 2  124 +/- 12  18 +/- 3
 333  9 +/- 2  5 +/- 3  27 +/- 8  156 +/- 29  18 +/- 2
 1000  9 +/- 2  7 +/- 3  22 +/- 12  155 +/- 6  20 +/- 8
 2000  4 +/- 2  13 +/- 4  16 +/- 3    
 3330  0 +/- 0a  0 +/- 0a  0 +/- 0a  0 +/- 0a  0 +/- 0a
 5000        0 +/- 0a  0 +/- 0a

Solvent control: 0.1 ml ethanol

1 The S9-mix contained 5% (v/v) S9 fraction

a Bacterial background lawn absent

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. Based on the results of this study it is concluded that unoxolTM 3,4 Dialdehyde is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Unoxol™ 3,4 Dialdehyde was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring

strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay

with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in

the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and β-naphthoflavone).

The study procedures described in this report were based on the most recent OECD and EEC guidelines.

Batch 200502771-6M of Unoxol™ 3,4 Dialdehyde was a clear to very pale-yellow liquid. The test substance was dissolved

in ethanol.

In the dose range finding test, Unoxol™ 3,4 Dialdehyde was tested up to concentrations of 5000 μg/plate in the absence and

presence of S9-mix in the strains TA100 and WP2uvrA. Unoxol™ 3,4 Dialdehyde did not precipitate on the plates at this

dose level. Cytotoxicity was evidenced by a complete lack of any microcolony background lawn at the dose levels of 3330

and 5000 μg/plate in both tester strains. Results for this dose range finding test were reported as part of the first experiment of

the mutation assay.

Based on the results of the dose range finding test, Unoxol™ 3,4 Dialdehyde was tested in the first mutation assay at a

concentration range of 33 to 3330 μg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537

and TA98. Cytotoxicity was evidenced by a complete lack of any microcolony background lawn in all three tester strains.

In an independent repeat of the assay with additional parameters, Unoxol™ 3,4 Dialdehyde was tested at a concentration

range of 10 to 2000 μg/plate in the absence S9-mix and at 10 to 3330 μg/plate in the presence of 10% (v/v) S9-mix in tester

strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Cytotoxicity was observed in all tester strains, except in tester strain

WP2uvrA in the absence of S9-mix.

Unoxol™ 3,4 Dialdehyde 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.

In this study, the negative and strain-specific positive control values were within our laboratory historical control data ranges

indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that Unoxol™ 3,4 Dialdehyde is not mutagenic in the Salmonella

typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.