Ethyl L-threoninate

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:

March 31, 2000 - April 19, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed in compliance with GLP standards.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

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:

other: Annex V of Directive 92/69/EEC (1993). B14 Mutagenicity Reverse Mutation Assays.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: UKEMS Guidelines (1990)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline (1997)

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Threoninethylester
- Analytical purity: 97%
- Purity test date: no data
- Lot/batch No.: 142-576-6
- Expiration date of the lot/batch: no data
- Stability under test conditions: considered to be stable under test conditions
- Storage condition of test material: frozen

Method

Target gene:

Strain Type of mutation in the histidine gene
TA 98 frame-shift
TA100 base-pair substitution
TA1535 base-pair substitution
TA1537 frame-shift
TA102 base-par substitution

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

NA

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Liver post-mitochondrial fraction (S-9) prepared from male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Concentration range in the range-finder experiment and mutation experiment (with and without metabolic activation): 1.6, 8, 40, 200, 1000 and 5000 µg/plate


Concentration range in Mutation experiment 2 (with and without metabolic activation): 156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate .

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

2-nitrofluorene was used without S9-mix at the following concentration: 5.0 µg/plate (TA98).

Controls 2

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Sodium azide was used without S9-mix at the following concentration: 2.0 µg/plate (TA100, TA1535).

Controls 3

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

9-aminoacridine was used without S9-mix at the following concentration: 50.0 µg/plate (TA1537).

Controls 4

Positive controls:

yes

Positive control substance:

other: glutaraldehyde

Remarks:

Glutaraldehyde was used without S9-mix at the following concentration: 25.0 µg/plate (TA102).

Controls 5

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Benzo[a]pyrene was used with S9-mix at the following concentration: 10.0 µg/plate (TA98).

Controls 6

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2-aminoanthracene was used with S9-mix at the following concentrations: 5.0 µg/plate (TA100, TA1535, TA1537) and 20.0 µg/plate (TA102).

Details on test system and experimental conditions:

METHOD OF APPLICATION:; direct plate incorporation method, and the preincubation method

DURATION
- Preincubation period: none in range-finder experiment and mutation experiment 1, 1 hour in the mutation experiment 2.
- Exposure duration: 3 days
- Expression time (cells in growth medium): NA
- Selection time (if incubation with a selection agent): NA
- Fixation time (start of exposure up to fixation or harvest of cells): NA

SELECTION AGENT (mutation assays): No data

NUMBER OF REPLICATIONS: triplicate plates

NUMBER OF CELLS EVALUATED: no data

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

OTHER EXAMINATIONS:
- Determination of polyploidy: NA
- Determination of endoreplication: NA
- Other: NA

Evaluation criteria:

Acceptance criteria:
1. the mean negative control counts fell within normal ranges
2. the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9 preparation.
3. no more than 5% of the plates were list through contamination or some other unforeseen event.

Evaluation criteria:
The test article was considered mutagenic if:
1. the assay was valid (see above)
2. Dunnett's test gave a significant response (p≤0.01) and the data set(s) showed a significant dose correlation
3. the positive responses described above were reproducible

Statistics:

The m-statistic was calculated to check that the data were Poisson-distributed, and Dunnett's test was used to compare the counts of each dose with control. The presence or ortherwise of a dose-response was checked by linear regression analysis.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: L-TEE was completely soluble in the aqueous assay system at all concentrations treated in each of the experiments performed.
- Precipitation: No precipitation was observed

RANGE-FINDING/SCREENING STUDIES: a range-finder experiment was performed.

COMPARISON WITH HISTORICAL CONTROL DATA: The solvent control and the positive controls were acceptable and fell within the normal historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed at any concentration.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

L-TEE was tested for mutation in five histine-required strains of Salmonella typhimurium, both in the absence and in the presence of metabolic activation S-9. All the test strains were tested at concentrations up to 5000 µg/plate as the maximum test dose. It was concluded that L-TEE did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), when tested under the conditions employed in this study.

Executive summary:

L-TEE was tested for mutation in five histidine-required strains of Salmonella typhimurium, both in the absence and in the presence of metabolic activation S-9. All the test strains were tested at concentrations up to 5000 µg/plate as the maximum test dose.

The mean number of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments. No L-TEE treatments of the test strains resulted in increased revertant numbers that could be considered indicative of any test article mutagenic activity. No signs of toxicity were apparent for any of the test plates, either with or without S-9.

It was concluded that L-TEE did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), when tested under the conditions employed in this study.