1,3-Isobenzofurandione, hexahydro-, reaction products with epichlorohydrin

Genetic toxicity: in vitro

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:

December 01, 1983 to December 19, 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study comparable to guideline study with acceptable retsriction.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only 4 strains of salmonella typhimurium tested (TA 98, TA100, TA1535 and TA1537). No E. coli or TA102.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

liquid

Method

Target gene:

Histidine dependence (auxotrophy)

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:

S9 fraction of liver from rats induced with Aroclor 1254

Test concentrations with justification for top dose:

20, 80, 320, 1280 and 5120 ug/plate

Vehicle / solvent:

Acetone

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

other: Daunorubicin-HCl (for TA 98), 4-nitroquinoline-N-oxide (for TA 100), N-methyl-N'-nitro-N-nitrosoguanidine (for TA1535) and Aminoacridine hydrochloride monohydrate (for TA 1537)

Remarks:

for the experiment without metabolic activation

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

for the experiment with metabolic activation Migrated to IUCLID6: on strain TA 1535

Details on test system and experimental conditions:

METHOD OF APPLICATION:
The substance was dissolved in acetone. Acetone alone was used for the negative controls (the substances and vehicles used for the positive controls are indicated below). Each Petri dish contains: 1) approx. 20 ml of minimum agar (Agar (Difco Laboratories, detroit, Michigan, USA plus salts (Vogel-Bonner Medium E) and glucose (Fluka, Buchs, Switzerland)), 2) 0.1 ml of a solution of the test substance or the vehicle and
0.1 ml of a bacterial culture (in nutrient broth, Difco Laboratories, detroit, Michigan, USA, 0.8% plus 0.5% NaCl) in 2.0 ml of soft agar. The soft agar is composed of: 100 ml of 0.6% agar solution with 0.6% NaCl and 10 ml of a solution of 1-histidine, 0.5 mM (Fluka, Buchs, Switzerland) and +biotin, 0.5 mM (Fluka, Buchs, Switzerland.).
In the experiments in which the substance is metabolically activated, 0.5 ml of an activation mixture is added also.
1 ml activation mixture contains: 0.3 ml S9 fraction of liver from rats (Tif:RAIf(SPF)) induced with Aroclor 1254 (Analabs.,Inc., North
Haven, Connecticut, U.S.A.) and 0.7 ml of a solution of cofactors.



DURATION
The plates were inverted and incubated for about 48 hours at 37°C +/- 1.5°C in darkness. Thereafter, the plates were evaluated by counting the colonies.

NUMBER OF REPLICATIONS:
Three plates per test substance concentration.

A confirmatory experiment was performed.

Evaluation criteria:

Doubling of the colony number with respect to the negative/solvent control at any concentration is evidence for mutagenicity

Statistics:

A statistical analysis was not performed.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

The substance exerted a clear-cut mutagenic action in this test system.

Executive summary:

The test material was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were

performed on strains TA 98, TA 100, TA 1535 and TA 1537 without and with microsomal activation with the following concentrations of the trial substance: 20, 80, 320, 1280 and 5120 ug/0.1 ml. In order to confirm the results, the experiments were repeated with the same concentration.

These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the

substances are demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone

back-mutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substances formed in mammals

would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat

liver microsomes and co-factors).

In the experiments performed without microsomal activation treatment with the test material led, to a distinct increase in the number

of back-mutant colonies of strain TA 1535 and TA 100 at the concentrations of 320, 1280 and 5120 ug/0.1 ml.

In the experiments with microsomal activation a distinct increase in the number of back-mutant colonies was observed with strain TA

100 at the concentrations of 80 ug/0.1 ml and above. With strain TA 1535 this effect was observed at all concentrations used.

The test material thus exerted a clear-cut mutagenic action in this test system.