1,2,3,6-tetrahydro-3-methylphthalic anhydride

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:

1995.12.8-1997.7.8

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1997

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Guidelines for screening Mutagenicity testing of Chemicals (Japan) and OECD Test Guideline 471 and 472

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrahydromethylphthalic anhydride

EC Number:

234-290-7

EC Name:

Tetrahydromethylphthalic anhydride

Cas Number:

11070-44-3

IUPAC Name:

3a-methyl-3a,4,5,6-tetrahydro-2-benzofuran-1,3-dione

Details on test material:

- Name of test material (as cited in study report): 1,3-isobenzofurandione, tetrahydromethyl (THMBF)
- Analytical purity: 99.97%
- Lot/batch No.: 2522
- Stability under test conditions: stable, purity 99.13% after testing
- Storage condition of test material: sealed, room temperature, dark place
- Molecular formula (if other than submission substance): C9 H10 O3
- Molecular weight (if other than submission substance): 166.18
- Smiles notation (if other than submission substance): O=C(OC(=O)C1CC=C(C2)C)C12
- InChl (if other than submission substance): 1/C9H10O3/c1-5-2-3-6-7(4-5)9(11)12-8(6)10/h2,6-7H,3-4H2,1H3
- Structural formula attached as image file (if other than submission substance): see Fig. (11070-44-3 structure.jpg)

Method

Target gene:

not indicated

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Type and identity of media: provided by Dr. A. N. Ames, California University, USA on 1975-10-31
- Properly maintained: yes, -80 °C
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: provided by Dr.Gada, National Institute of Genetics, Japan on 1979-5-9
- Properly maintained: yes, -80 °C
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

-S9 mix; 0, 62.5, 125, 250, 500, 1000, 2000 ug/plate
+S9 mix; 0, 156, 313, 625, 1250, 2500, 5000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: well-known solvent for general purpose

Details on test system and experimental conditions:

Type: Ames test
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk


DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:2

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: The test substance is considered to be positive for mutagenic activity when assay plates with the test substance show a significant increase in revertant colony count as compared with that on negative control plates and when this effect is reasonably reproducible or dose dependent.

Evaluation criteria:

no details given

Statistics:

no details given

Results and discussion

Test results

Species / strain:

other: Salmonella typhimurium, TA100, TA1535, TA98, TA1537, Escherichia coli Wp2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: without metabolic activation(-S9mix)| 500 ug/plate (TA1535),| 1000 ug/plate (TA100, TA98, TA1537),| 2500 ug/plate (WP2)|with metabolic activation(+S9mix)| 5000 ug/plate (TA100, TA1537)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

This chemical did not induce mutations in the S. typhimurium and E. coli strains.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 7.4

Remarks on result:

other: other: Salmonella typhimurium, TA100, TA1535, TA98, TA1537, Escherichia coli Wp2 uvrA

Any other information on results incl. tables

no remarks

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Tetrahydromethylphthalic anhydride (MTHPA) was not mutagenic in Salmonella typhimuriumTA100, TA1535, TA98, TA1537 and Escherichia coli WP2uvrA at concentrations up to 5 mg/plate or 2 mg/plate, with or without an exogenous metabolic activation system, respectively.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA100, TA1535, TA98, TA1537 of S. typhimurium and Escherichia coli Wp2 uvrA were exposed to tetrahydromethylphthalic anhydride in DMSO at concentrations of 0, 62.5, 125, 250, 500, 1000, 2000 ug/plate (-S9 mix) and 0, 156, 313, 625, 1250, 2500, 5000 ug/plate (+S9 mix) in the presence and absence of mammalian metabolic activation (S9 mix, Rat liver, induced with Phenobarbital and 5,6-benzoflavone).

Tetrahydromethylphthalic anhydride was tested up to limit concentration (5000 µg/plate). No cytotoxicity occurred. The test chemical did not induce mutations in the S. typhimurium and E. coli strains The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for the Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals Test Guideline and OECD 471 and 472 for in vitro mutagenicity (bacterial reverse gene mutation) data.