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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:
weight of evidence
Study period:
1991-09-03 to 1991-09-05
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study report does not include a GLP compliance certificate and it does not indicate any specific OECD guidelines. However, the report indicates that the study was conducted according to the appropriate OECD guidelines and the study primarily followed the methodology described in the corresponding OECD guideline number 471. The methodological deficiencies identified in the report were not considered to adversely affect the validity of the study.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
: no indication of dose levels used in body of the report. There is no indication in the corresponding OECD guideline (471) that the strain TA1538 is appropriate to use.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
: analyses to determine the uniformity, concentration, or stability of the test or control mixtures were not performed by the testing facility.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
: 2-Aminoanthracene was used as the sole indicator of S-9 efficacy
Principles of method if other than guideline:
The methodological deficiencies were not considered to have affected the overall outcome of the study.
GLP compliance:
yes
Remarks:
: self-certified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): trimellitic anhydride
- Physical state: White flakes
- Lot/batch No.: FSF-375
- Storage condition of test material: Stored at room temperature and protected from exposure to light.

Method

Target gene:
Histidine mutations hisG46, hisC3076, hisD3052.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: A mutation in the histidine operon. The rfa wall mutation causes a loss of one of the enzymes responsible for the synthesis part of the lipopolysaccharide layer of the cells wall. A deletion in the uvrB gene resulting in a deficient DNA excision-repair
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: A mutation in the histidine operon. The rfa wall mutation causes a loss of one of the enzymes responsible for the synthesis part of the lipopolysaccharide layer of the cells wall. A deletion in the uvrB gene resulting in a deficient DNA excision-repair
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
0, 33, 100, 333, 1000, 3333 and 10000 µg/plate.
Vehicle / solvent:
DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
1.0 µg/plate
Positive control substance:
sodium azide
Remarks:
without metabolic activation for TA 100 and TA 1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
1.0 µg/plate
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation for TA 98 and TA1538
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
75 µg/plate
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation for TA 1538
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
1.0 µg/plate
Positive control substance:
other: 2-Aminoanthracene
Remarks:
with metabolic activation for TA 98, TA 100, TA 1535, TA 1537 and TA 1538
Details on test system and experimental conditions:
The tester strains were histidine auxotrophs. They were received directly from Dr. Ames, Department of Biochemistry, University of California.
Frozen permanent stocks were prepared by growing fresh overnight cultures, adding dimethylsulfoxide and freezing 1.5 ml aliquots. The frozen permanent stocks were stored at or below 70°C. Master plates were prepared by streaking each tester strain from a frozen permanent onto minimal medium supplied with histidine and biotin, and for strains containing the R-factor, ampicillin. Master plates were stored at 4±2°C.

Overnight cultures were prepared by removing a colony of the appropriate strain from the master plate and transferring it to a vessel containing ~50 ml culture medium. The length of incubation was controlled and monitored to ensure that cultures were harvested in late log phase. Following inoculation, each flask was placed in a resting shaker/incubator at room temperature. The shaker/incubator was programmed to begin shaking at approximately 125 rpm at 37±2°C approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titre of approximately 1-2x10E9 cells/ml.
On the day of their use in the mutagenicity assay, all tester strain cultures were checked for the correct genotype. The presence of the rfa wall mutation was confirmed by demonstration of sensitivity to UV light. The presence of the pKM101 plasmid was confirmed by demonstration of resistance to ampicillin. Spontaneous reversion frequencies in the vehicle controls were demonstrated by plating 100 µl aliquots along with the appropriate vehicle on selective media.

Dosing solutions were not adjusted to compensate for the purity of the test substance. A dose range-finding study was conducted, the maximum dose tested was 10000 µg/plate.

Revertant colonies were counted either by automated colony counter or by hand.
Evaluation criteria:
A test article was considered positive if it caused a dose-related increase in the mean revertants per plate of at least one strain with a minimum of two increasing concentrations.
Statistics:
Formal statistical analysis were not carried out; the mean revertants per plate and the standard deviation was calculated.

Results and discussion

Test resultsopen allclose all
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the dose range-finding study, the maximum dose tested (10000 µg/plate) did not precipitate, but toxicity was observed. This dose was chosen as the top dose for the mutagenicity study.

In the mutagenicity assays, for tester strains TA 98, TA 1535 and TA 1538, no positive responses were observed in the presence of microsomal enzymes and with any of the tester strains in the absence of microsomal enzymes. A 2.0 fold, non-dose responsive increase was observed with tester strain TA 1538 in the absence of microsomal enzymes. However, non-dose responsive increases are not evaluated as positive. No positive response was observed with tester strain TA 100 in the presence of microsomal enzymes. No positive response was observed with tester strain TA 1537 in the presence of microsomal enzymes.

In the confirmatory assay, no positive responses were observed with any of the tester strains in the presence and absence of microsomal enzymes.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

No increase in the numbers of revertant colonies were seen following exposure to the test material.

Applicant's summary and conclusion

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

Under the conditions of this study, trimellitic anhydride did not cause a positive response with any of the tester strains in the presence and absence of microsomal enzymes.
Executive summary:

Trimellitic anhydride was assessed for mutagenicity using Salmonella tester strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 in the presence and absence of rat liver microsomal enzymes using the plate incorporation technique. A dose range-finding study was conducted to establish the dose range to be used in the mutagenicity and confirmatory assays.

Based on the results of the dose-range finding study, the maximum dose tested in the mutagenicity and confirmatory assays was 10000 µg per plate. No increase in the numbers of revertant colonies were seen following exposure to the test material. Appropriate positive control compounds confirmed the sensitivity of the assay.

The results of this assay indicate that under the conditions of this study, the test article did not cause a positive response in the Salmonella/Mammalian-Microsome Plate Incorporation Mutagenicity Assay.