Tetrahydrophthalic anhydride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2016-09-13 to 2016-10-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrahydrophthalic anhydride

EC Number:

247-570-9

EC Name:

Tetrahydrophthalic anhydride

Cas Number:

26266-63-7

Molecular formula:

C8H8O3

IUPAC Name:

tetrahydro-2-benzofuran-1,3-dione

Test material form:

solid - liquid: suspension

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Sample from production lot
- Lot/batch No.of test material: Lab sample of October 9th 2015
- Expiration date of the lot/batch: 2016-10-08
- Purity: 99.91%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient conditions
- Solubility and stability of the test substance in the solvent/vehicle: Soluble

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Mixed induction rat liver S9

Test concentrations with justification for top dose:

19.5 - 5000 micrograms/plate. Highest concentration evaluated is that indicated in the test guidelines

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Type and identity of media:
- The following growth media were used:
- Nutrient Broth: Oxoid Nutrient Broth No 2 used for the preparation of liquid cultures of the tester strains.
- Nutrient Agar: Oxoid Nutrient Broth No 2 and Difco Bacto-agar plates were used for the non-selective growth of the tester strains.
- Minimal Agar: Minimal medium agar was Difco Bacto-agar in Vogel Bonner Medium E, with 2% Glucose
- Top Agar: was 0.6% Difco Bacto-agar + 0.5% NaCl wsith 0.5 mM Biotin + 0.5 mM Histidine (S. typhimurium tester strains) or 0.5 mM tryptophan (E. Coli tester strain) added to the agar.

DURATION
- Preincubation period: 72 hours at +37°C

NUMBER OF REPLICATIONS:3 replicate for each point

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only.
In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

Regression analysis to fit a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None
- Precipitation: No

Applicant's summary and conclusion

Conclusions:

IThe tested substance, THPA, does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism.

Executive summary:

Gene mutation has been investigated in bacteria using strains of Salmonella typhimurium and Escherichia coli, in accordance with OECD/EU test methods. Five tester strains TA1535, TA1537, TA98, TA100

and WP2uvrA were used and experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and

betanaphthoflavone. The tested substance, tetrahydrophthalic anhydride (THPA), did not induce reverse mutation in the tester strains, neither in the absence nor presence of S9 metabolism.