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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
The substance is negative (non-genotoxic) in bacterial mutagenicity and mammalian cell clastogenicity studies.
Link to relevant study records
in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study according to GLP
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
according to guideline
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254 was prepared according to MARON and AMES (1983) at LPT.
Test concentrations with justification for top dose:
3.16, 10.0, 31.6, 100, 316 and 1000 µg per plate, 3 plates per concentration.
Vehicle / solvent:
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
sodium azide
other: 2-aminoanthracene
see details on test system and conditions for concentrations of positive control substances.
Details on test system and experimental conditions:
1st independent experiment - Plate Incorporation Method:
Sterile top agar containing 0.6% agar and 0.5% NaCl was molten on the day of the test. 10 mL of a sterile solution of 0.5 mM L-histidine HCl/0.5 mM biotin were added to 100 mL of molten agar. 2 mL of this top agar were distributed into culture tubes held at 45°C in a heating block. 0.1 mL of Salmonella cell suspension (containing approximately 108 viable cells in the late exponential or early stationary phase), 0.1 mL of test item solution (or 0.1 mL solvent or 0.1 mL positive control) and 0.5 mL of S9 mix were added to these culture tubes. In the assay without metabolic activation, the S9 mix was substituted with 0.5 mL phosphate buffer mentioned above.
The test components were mixed by vortexing the soft agar for 3 sec at low speed and then poured onto a coded 27.5 mL minimal glucose agar plate (Minimal Glucose Agar medium E)). To achieve a uniform distribution of the top agar on the surface of the plate, the uncovered plate was quickly tilted and rotated and then placed on a level surface with the cover on and finally allowed to harden.
Immediately, the plates were inverted and placed in a dark 37°C incubator for 48 to 72 hours. The revertant colonies on the test plates and on the control plates were counted with a colony counter , and the presence of the background lawn on all plates was confirmed.

2nd independent experiment - Preincubation Method
The test item/test solution was preincubated with the test strain (containing approximately 108 viable cells in the late exponential or early stationary phase) and sterile buffer (0.5 mL) or the metabolic activation system (0.5 mL) for 20 minutes at 37°C prior to mixing with the overlay agar and pouring onto the surface of a minimal agar plate. 0.1 mL of the test item/test solution, 0.1 mL of bacteria, and 0.5 mL of S9 mix or sterile buffer, were mixed with 2 mL of overlay agar. Tubes were aerated during preincubation by using a shaker. The remaining steps were the same as described for the plate incorporation method.

Concentration of positive control substances:
Sodium azide: 10 µg/plate, in water
2-nitrofluorene, 10 µg/plate, in DMSO
9-aminoacridine, 100 µg/plate, in absolute ethanol
methylmethane sulfonate, 1300 µg/plate, in DMSO
cyclophosphamide, 1500 µg/plate, in water
2-aminoanthracene, 2 µg/plate, in DMSO

Vehicle controls were undertaken; there were no true negative controls.

Evaluation criteria:
a test item is considered to show a positive response if
- at one or more concentrations the number of revertants is reproducibly increased in at least one strain with or without metabolic activation. A 2-fold increase in comparison to the solvent control is regarded as being relevant for a positive response in the strains TA 98, TA 100 and TA 102. For the strains TA 1535 and TA 1537 a 3-fold increase represents a biological relevant effect.
- a concentration-related increase of the revertants is observed. A 2-fold increase in comparison to the solvent control is regarded as being relevant for a positive response in the strains TA 98, TA 100 and TA 102. For the strains TA 1535 and TA 1537 a 3-fold increase represents a biological relevant effect.
- Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
- Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the solvent control and/or a scarce background lawn.
The Mann and Whitney test (p ≤ 0.05, see section 6, reference 3.) may be used to determine statistical significance. The Spearman's rank correlation coefficient (section 6, reference 3.) may also be applied.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
Scarce background lawn and reduction of the number of revertants was noted at the top concentration of 1000 µg/plate in all test strains.
Vehicle controls validity:
Untreated negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
No increase in revertant colony numbers as compared with control counts was observed for 3,3’,4,4’-benzophenonetetracarboxylic dianhydride (BTDA), tested up to a cytotoxic concentration of 1000 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).
The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.
Interpretation of results (migrated information):
negative with and without metabolic activation

1,3-Isobenzofurandione, 5,5'-carbonylbis- (BTDA) was tested in an OECD 471 bacterial reverse mutation assay of Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, using both the plate incorporation method and the preincubation method. Either with and without metabolic activation (rat liver S9 fraction from Arochlor-treated male rats), the test material did not cause an increase in the number of revertants of any strain, up to a cytotoxic concentration of 1000 µg/plate. The substance is not mutagenic under conditions of this assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

An OECD 471 bacterial reverse mutation assay (using both the plate incorporation and preincubation protocols) was undertaken, under conditions with and without S9 metabolic activation from livers of Arochlor 1254-induced male rats, to assess the in-vitro genotoxicity of BTDA. Additionally, BTDA was tested in an OECD 472 unscheduled DNA repair assay in primary hepatocytes (metabolically-competent) isolated from male Fisher 344 rats. There was no increase in the incidence of bacterial mutation or DNA repair at concentrations of BTDA up to cytotoxic doses.

An in vivo assay for chromosome aberrations was also undertaken in CD rats administered powdered BTDA via the inhalation route (concentrations of 0, 1, 55 and 3000 mg/m3 inhalable size particles, via whole body exposure for 6 hours per day, 5 days per week for 1 week). Mortality occurred only at the high dose of 3000 mg/m3. Triethylene melamine was given intraperitoneally as the cytogenetic positive control. Cytogenetic findings were negative at all concentrations, supporting the conclusion that BTDA is not genotoxic.

Justification for selection of genetic toxicity endpoint
Experimental results

Justification for classification or non-classification

Experimental evidence supports the conclusion that the substance is not mutagenic and thus does not meet the criteria for classification and labeling as a mutagen, according to Regulation EC No. 1272/2008.