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Genetic toxicity in vitro

Description of key information

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

Link to relevant study records

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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
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
Target gene:
hisD3052, hisG46, hisG428 (paQ1), hisC3076
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
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)
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

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.

Conclusions:
Trimethyl orthoformate is not a bacterial mutagen.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
not explicitly stated
Deviations:
yes
Remarks:
Only strains TA97, TA98; TA100
GLP compliance:
yes
Remarks:
Quality assurance statement attached
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine gene, frame-shift or base-pair substitution
Species / strain / cell type:
S. typhimurium TA 97
Additional strain / cell type characteristics:
other: rfa character, presence of pKM101
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: rfa character, presence of pKM101
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: rfa character, presence of pKM101
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 fraction induced with Aroclor-1254
Test concentrations with justification for top dose:
8, 40, 200, 1000, 5000 microgram/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not stated
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
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
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: at least 2 days

NUMBER OF REPLICATIONS: triplicate; negative and positive controls quintuplicate

NUMBER OF CELLS EVALUATED: not stated

DETERMINATION OF CYTOTOXICITY
- Method: other: inspection of the bacterial background lawn

Evaluation criteria:
VALIDITY:
- Mean negative control counts within normal range (from historical solvent control values, tabulated in the report)
- Positive control chemicals induce clear increases in revertant numbers, confirming discrimination between different strains, and an active S9 preparation
- No more than 5% of the plates lost (through contamination or other cause); at no data point: all replicates lost

EVALUATION: A test compound was considered to be mutagenic if
- The assay was valid
- There were two-fold increases in revertant numbers, accompanied by significant F-statistics and dose-response correlations
Statistics:
- Mean and standard deviation of the plate counts for each treatment
- Analysis of variance (F-test) and regression analysis, if a two-fold (or greater) increase in revertants over the solvent control is observed
Species / strain:
S. typhimurium TA 97
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 98
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 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
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:

RANGE-FINDING/SCREENING STUDIES: Pretest (plate-incorporation) with strain TA 100 showed no signs of toxicity (with and without S9)

COMPARISON WITH HISTORICAL CONTROL DATA: Ranges for spontaneous revertants on solvent control plates from a large volume of historical control data tabulated in report and used for valididy assessment

ADDITIONAL INFORMATION ON CYTOTOXICITY: No signs of reduced growth of the bacterial background lawn, indicative of test compound induced cytotoxicity
Remarks on result:
other: all strains/cell types tested

Maximum increase of revertants over controls: 0.9 - 1.2 without metabolic activation, 1.0 - 1.3 with metabolic activation (in all strains)

Additional information

Ames test (bacterial reverse mutation in vitro): Negative

A key study conducted in 2002 according to guideline OECD 471 and under GLP is available. The study is considered to be relevant, reliable (Klimisch 1) and adequate for the purposes of risk assessment, classification and labelling. Five S. typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 were investigated; TA102 is listed as a valid alternative to E. coli WP2 uvrA or E. coli WP2 uvrA (pKM101), so that the requirements of the guideline are fully met. Trimethyl orthoformate did not elicit a mutagenic effect in any of the strains, with and without metabolic activation, and is therefore not considered a bacterial mutagen.

A supporting study (on 18 test substances, GLP, 1987) investigated only three S. typhimurium strains (TA97, TA98, TA 100), none of which showed any mutagenic response.

Chromosome aberration: No data available


Short description of key information:
Ames test (Infracor, Enste-Diefenbach 2002): negative (S. typhimurium TA98, TA100, TA102, TA1535, TA1537)
Ames test (Microtest, Kenelly 1987): negative (S. typhimurium TA97, TA98, TA100)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Both in vivo studies considered were negative and therefore do not warrant classification.