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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD)

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
2,5,6-trimethylcyclohex-2-en-1-one
EC Number:
243-473-0
EC Name:
2,5,6-trimethylcyclohex-2-en-1-one
Cas Number:
20030-30-2
Molecular formula:
C9H14O
IUPAC Name:
2,5,6-trimethylcyclohex-2-en-1-one
Details on test material:
- Name of test material (as cited in study report): 2,3,6-Trimethylcyclohexen-1-on
- ZST Test Substance No . : 87/818
- Analytical purity: ca. 96 %
- Storage condition of test material: room temperature

Method

Species / strain
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 S9 mix
Test concentrations with justification for top dose:
Standard plate test (SPT): 100, 500, 2500 and 5000 ug/plate
Preincubation test (PIT): 100, 500 and 2500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 10 µg/plate, dissolved in DMSO, for the strains TA100, TA98, TA1537 and TA1535
Remarks:
with metabolic activation
Positive controls:
yes
Positive control substance:
other: N-methyl-N-nitro-N-nitrosoguanidine; 5 µg/plate, dissolved in DMSO, for strains TA100 and TA1535
Remarks:
without metabolic activation
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine, 10 µg/plate, dissolved in DMSO, for strain TA98
Remarks:
without metabolic activation
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation

Migrated to IUCLID6: 100 µg/palte, dissolved in DMSO, for strain TA1537
Details on test system and experimental conditions:
METHOD OF APPLICATION:
The experimental procedures are based on the methods described by Ames, (1973, 1975), Yahagi et al. (1977) and Matsushima et al. (1980).

1ST EXPERIMENT: in agar (plate incorporation) (SPT)

DURATION
- Exposure duration: 48 - 72 h, 37°C


2ND EXPERIMENT: preincubation (PIT)


DURATION
- Preincubation period: 20 min, 37°C
- Exposure duration: 48 - 72 h, 37°C

NUMBER OF REPLICATIONS: triplicates; 3 test plates per dose or per control


DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp- background growth), reduction in the titer
Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
depending on the strain and test conditions at doses > 2500 µg/plate .
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Results of SPT:

Strain metabolic activation Experiment  controls µg substance / plate Evaluation
solvent positive 20 100 500 2500 5000
TA 1535 - 1 17 2100 15 17 16 11 11 -
- 2 12 2150 22 18 23 13 8 -
- 3 14 1950 20 14 10 18 7 -
+ 1 21 123 21 23 19 19 7 -
+ 2 19 159 17 18 20 20 8 -
+ 3 19 154 22 18 23 16 13 -
TA 1537 - 1 7 245 8 6 11 8 B -
- 2 8 372 5 7 7 6 B -
- 3 8 367 8 10 8 9 B -
+ 1 12 129 6 7 9 7 7 -
+ 2 9 117 9 14 11 9 8 -
+ 3 8 142 8 8 8 9 4 -
TA 98 - 1 21 575 20 21 23 22 13B -
- 2 22 647 19 23 26 21 10B -
- 3 24 652 26 18 20 16 13B -
+ 1 33 1310 42 33 36 32 28 -
+ 2 32 1180 31 49 38 36 28 -
+ 3 35 1060 31 35 41 38 33 -
TA 100 - 1 101 1820 106 97 120 98 60B -
- 2 97 1920 97 101 118 101 71B -
- 3 102 1950 120 98 101 91 70B -
+ 1 121 1890 135 122 111 102 97 -
+ 2 128 1830 105 133 103 101 84 -
+ 3 115 1870 119 133 128 114 88 -

B: reduced his-backround

Results of PIT:

mean number of revertant colonies / plate  
Strain metabolic activation Experiment  controls µg substance / plate Evaluation
solvent positive 4 20 100 500 2500
TA 1535 - 1 19 1050 21 17 13 18 13 -
- 2 12 1080 21 15 13 16 13 -
- 3 12 1100 13 15 13 14 13 -
+ 1 13 139 15 17 18 12 13 -
+ 2 16 132 19 18 20 12 13 -
+ 3 18 110 22 19 17 14 13 -
TA 1537 - 1 8 423 7 11 10 5 B -
- 2 8 413 9 7 8 3 B -
- 3 7 377 9 10 7 5 B -
+ 1 13 129 9 8 7 8 B -
+ 2 12 100 9 8 9 10 B -
+ 3 11 85 9 8 10 11 B -
TA 98 - 1 19 985 24 26 26 23 B -
- 2 22 1050 26 23 21 13 B -
- 3 20 1330 24 21 20 9 B -
+ 1 32 878 30 41 33 33 B -
+ 2 36 810 34 30 30 34 B -
+ 3 34 700 37 34 40 36 B -
TA 100 - 1 106 1160 128 115 110 93 B -
- 2 106 1170 107 127 86 89 B -
- 3 118 1100 106 107 103 112 B -
+ 1 112 1060 123 98 118 128 B -
+ 2 128 1150 104 106 107 134 B -
+ 3 112 920 115 126 133 124 B -

B: reduced his-backround

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

According to the results of the present study, the test substance 2,3,6-Trimethylcyclohexen- 1-on is not mutagenic in the Ames test under the experimental conditions chosen above.

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