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Toxicological information

Genetic toxicity: in vitro

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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:
2002-02-14 to 2002-05-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Remarks:
S. typhimurium TA102 is a valid alternative to E. coli WP2 uvrA
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Trimethylorthoformate
- Substance type: pure active substance
- Physical state: clear liquid
- Analytical purity: 99.5 % (determined by sponsor)
- Impurities (identity and concentrations): no data
- Lot/batch No.: 11029 M, manufactured 2001-10-29
- Expiration date of the lot/batch: 2002-10-29
- Stability under test conditions: Not tested

Method

Target gene:
hisD3052, hisG46, hisG428 (paQ1), hisC3076
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: contains R-factor plasmid pKM101
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction (lot # 270701, RCC Cytotest Cell Research , Rossdorf, Germany) induced with phenobarbital + beta-naphthoflavone
Test concentrations with justification for top dose:
Test #AM-02/04.1: Plate incorporation test: 50, 160, 500, 1600, 5000 microgram/plate
Test #AM-02/04.2: Preincubation test: 50, 160, 500, 1600, 5000 microgram/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: miscible with test substance, compatible with the survival of the bacteria and S9 activity
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminofluorene and 2-aminoanthracene for all strains
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: Test #AM-02/04.1: in agar (plate incorporation), Test #AM-02/04.2: preincubation

DURATION
- Preincubation period: 30 min
- Exposure duration: 72 hours at 37°

NUMBER OF REPLICATIONS: Triplicate, per strain and concentration of the test material

NUMBER OF CELLS EVALUATED: >7'100'000 per plate

DETERMINATION OF CYTOTOXICITY
- other: pre-experiment for toxicity (with strains TA 98 and TA 100) with seven concentrations (25 - 5000 microgram/plate) showed no signs of toxicity (reduction of background lawn)

Evaluation criteria:
VALIDITY:
- Characteristic mean number of spontaneous revertants (of each strain) in solvent control
- Titers of overnight cultures > 100'000'000
- Significant increase in the number of revertants in the mean of each positive control, compared with the mean of the solvent control; exception: in case of no significant increase of revertants with 2-aminofluorene, a parallel significant increase with 2-aminoanthracene will be regarded as sufficient, and vice versa.
- At least four non-toxic dose levels

EVALUATION:
- At least a doubling in the mean revertants per plate of at least one tester strain (in two independent experiments)
- This increase must be accompanied by a dose response to increasing concentrations of the test article
- Single increases in revertant frequencies (not dose-related, not reproducible in independent tests) are considered non-relevant
- If such increases occur in both independent tests, this will be taken as an indication of a mutagenic effect
Statistics:
Mean and standard deviation of replications
Test compound / control ratio: Mean no. of colonies/plate (test compound) / mean no. of colonies/plate (water)

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
other: Positive controls valid, except 2-aminoanthracene; parallel 2-aminofluorene showed enough activity, therefore, condition for validity was met
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not tested
- Effects of osmolality: not tested
- Evaporation from medium: not tested
- Water solubility: fully miscible, no effect
- Precipitation: not observed
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: Pretest (plate-incorporation test with strains TA 98 and TA 100 showed no toxicity and no induction of mutation.

COMPARISON WITH HISTORICAL CONTROL DATA: Based on tests performed 1998 - 2001 (TA 102: 1999 and 2001), only insignificant deviations were observed (one test higher, several tests lower). The results were regarded as acceptable.

ADDITIONAL INFORMATION ON CYTOTOXICITY: A reduced growth of the bacterial background lawn, indicative of test compound induced cytotoxicity, was not detectable with any of the tester strains.
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

In both experiments (test #AM-02/04.1: plate incorporation test; test #AM-02/04.2: preincubation test), the treatment with the test compound did not result in a dose-related significant increase in the revertant frequency in any of the five tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, with or without metabolic activation by S9 mix.

All criteria for a valid study were met as described: All tested bacterial strains exhibited a positive mutagenic response with the positive controls (with and without metabolic activation; TA 102: only with 2-aminofluorene). Solvent controls (water) were also tested with each strain, and the mean numbers of spontaneous revertants were considered acceptable.

Trimethyl orthoformate did not induce a mutagenic effect in S. typhimurium, and is therefore not considered a bacterial mutagen.

Applicant's summary and conclusion

Conclusions:
Trimethyl orthoformate is not a bacterial mutagen.