Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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 resultsopen allclose all
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
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
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
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
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.