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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
An in vitro gene mutation study in bacteria is a standard information requirement in Annex VII to REACH. The study meets the requirements of OECD TG 471 (1996) because it was performed with 5 strains: four strains of S. typhimurium (TA98; TA100; TA1535; TA1537) and another strain that is E. coli WP2 uvrA.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

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

Test material

Constituent 1
Reference substance name:
Isodecyl benzoate
IUPAC Name:
Isodecyl benzoate

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver preparations
Test concentrations with justification for top dose:
0, 312.5, 625, 1250, 2500, 5000 ug/ plate. No toxicity observed at 5000 ug/plate
Vehicle / solvent:
DMSO
Controlsopen allclose all
Positive controls:
yes
Positive control substance:
other: In presence of S-9 mix: 2-Aminoanthracene
Remarks:
2ug/plate (TA 1535/1537), 0.5 ug/plate (TA 98), 1ug/plate (TA 100), 10ug/plate (WP2 uvrA)
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Absence of S-9 mix - 1 ug/plate for strain TA 98
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Absence of S-9 mix - 80 ug/plate for TA 1537
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
N-ethyl-N-nitro-N-nitroguanidine 5ug/plate (TA 1535), 3ug/plate (TA 100) , 2 ug/plate (WP2 uvrA)
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Absence of S-9 mix - N-ethyl-N-nitro-N-nitroguanidine 5ug/plate (TA 1535), 3ug/plate (TA 100) , 2 ug/plate (WP2 uvrA)
Details on test system and experimental conditions:
PRELIMINARY TOXICITY TEST
Four concentrations of test substance were assessed for toxicity using the five tester strains. The
highest concentration was 50 mg/mL of test substance in the hosen solvent, which provided final
concentration of 5000 ug/plate. Three 10-fold serial dilutions of the highest concentration were also
tested. The chosen sołvent, dimethyl sulphoxide, was used as the negative control.
An aliquot of 0.I ml of a 10 hour bacterial culture and 0.5 ml S-9 nix or 0.5 mi 0.1 M phosphate
buffer (pH 7.4) were placed in glass bottles. An aliquot of 0.1 ml of the test solation was added,
followed immediately by 2 ml of histidine/tryptophan deficient agar. The mixture was thoroughly
shaken and overlaid onto previously prepared petri dishes containing 25 mi minimal agar. A single
petri dish was used for each dose level. Plates were also prepared without the addition of bacteria
in order to assess the sterility of the test substance, S-9 mix and phosphate buffer, All plates were
incubated at 37"C for 3 days. After this period the appearance of the background bacterial lawn was
examined. Revertant colonies were counted using a Seescan Automatic Colony Counter
Any toxic effects of the test substance can be detected by a substantial reduction in revertant colony
counts or by the absence of a complete background bacterial lawn. In the absence of any toxic effects
the top concentration used in the main tests is the same as that used in the preliminary toxicity test.
If toxic effects are observed a lower concentration may be chosen for the main assays. ideally the
concentrations chosen for the mutation tests should include a minimum of four non-toxic
concentrations.

MUTATION TEST PROCEDURE
The test substance was added to cultures of the five tester strains at five concentrations separated by
2-4 fold dilutions. The highest concentration of isodecyl benzoate used was S000 plate. The negative control was the chosen solvent, dimethyl sulphoxide The positive control compounds were
also included.

An aliquot of 0. ml of a 10 hour bacterial culture and 0.5 mi S9 mix or 0.5 ml 0,.1 M phosphate
buffer (pH 7.4) were placed in glass bottles, An aliquot of 0.I al of the test solution wis added,
followed immediately by 2 ml of histidine/tryptophan deficient agar. The mixture was thoroughly
shaken and overlaid onto previously prepared petri dishes containing 25 ml minimal agar, Three petri
dishes were used for each dose level. A set of plates were also prepared containing only bacterial
culture and S9 mix or phosphate buffer (0 ug/plate). Plates were also prepared without the addition
of bacteria in order to assess the sterility of the test substance, S-9 mix and phosphate buffer, All
plates were incubated at 37C for 3 days. After this period revertant colonies were counted using
a Seescan Automatic Colony Counter.

At a later date the main test was repeated using the procedure described above with the same
concentrations of test substance

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Isodecyl benzoate is not mutagenic.
Executive summary:

The revertant colony counts for isodecyl benzoate obtained in the preliminary toxicity test are showed
chosen as the top dose level in the mutation tests. Isodecyl benzoate was not toxic towards the tested strains. Therefore 5000 ug/plate was chosen as the top dose level in the mutation tests.



No substantial increases in revertant colony numbers of any of the tester strains were observed
following treatment with isodecyl bezoate at any dose level, in the presence or abscence of S-9 mix,
in either mutation test.



The concurrent positive control compounds demonstrated the sensitivity of the say and he
metabolizing activity of the liver preparations.



CONCLUSION
It is concluded that, when tested in dimethyl sulphoxide, Isodecyl benzoate shows no evidence of
mutagenic activity in these bacterial systems.