Methyl L-threoninate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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 5000µg/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 (OECD 471).

L-TME holds the same structure as L-TEE except that L-TME contains a methyl alkyl-side group to the ester bond and L-TEE an ethyl alkyl-side group. The toxicokinetic profile of L-TME and L-TEE is different in terms of the alcohol part (methanol and ethanol). For mutagenic activity, hydrolysis of the ester bond is expected liberating alcohols where methanol is considered to be more toxic than ethanol. However, methanol is shown non-mutagenic in Ames test. Due to the structural similarity to L-TEE (the L-Threonine part) and the differences in toxicokinetic properties (methanol and ethanol), the mutagenic activity of L-TME is described by the toxicity of methanol.

Due to read across to negative mutagenicity data on L-TEE and further considering the negative mutagenicity data on methanol, L-TME is considered to be non-mutagenic.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

L-TME holds the same structure as L-TEE except that L-TME contains a methyl alkyl-side group to the ester bond and L-TEE an ethyl alkyl-side group. The toxicokinetic profile of L-TME and L-TEE is different in terms of the alcohol part (methanol and ethanol). For mutagenic activity, hydrolysis of the ester bond is expected liberating alcohols where methanol is considered to be more toxic than ethanol. Due to the structural similarity to L-TEE (the L-Threonine part) and the differences in toxicokinetic properties (methanol and ethanol), the mutagenic activity of L-TME is described by the toxicity of methanol. Due to read across to negative mutagenicity data on L-TEE and further considering the negative mutagenicity data on methanol, L-TME is considered to be non-mutagenic. Further information on read across to L-TEE using the analogue approach can be found in the data matrix table attached as background material and in section 13.

Justification for type of information:

Data on target substance is not available. Thus, read-across has been applied using data from the source substance L-Threonine Ethyl Ester (L-TEE). See further read-across justification in attached background material and in section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UKEMS Guidelines (1990)

Deviations:

no

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

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 / 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

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).

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).

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).

Positive controls:

yes

Positive control substance:

other: glutaraldehyde

Remarks:

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

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).

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.

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.

Remarks on result:

other: all strains/cell types tested

The toxicokinetic properties of L-TEE and L-TME are evaluated to be different. Read across to L-TEE is possible for endpoints in which hydrolysis of the ester bond does not take place, e.g. skin irritation and sensitisation. For endpoints in which hydrolysis takes place, thereby liberating the alcohols, i.e. methanol in the case of L-TME and ethanol in the case of L-TEE, the toxicities of the hydrolysis products must be taken into account. For mutagenic activity, hydrolysis of the ester bond is expected liberating alcohols where methanol is considered to be more toxic than ethanol.

Testing data (OECD 471, Supporting study, Klimisch score 2) retrieved from the REACH registration dossier of methanol (CAS No 67-56-1; EC No 200-659-6) shows that methanol is non-mutagenic in the Ames test (+/- S9). Further, testing data on L-TEE (OECD 471) shows no mutagenic activity in the Ames test (+/- S9), hence L-threonine part of L-TME is non-mutagenic.

Due to read across to negative mutagenicity data on L-TEE and further considering the negative mutagenicity data on methanol, L-TME is considered to be non-mutagenic.

Further information on the analogue approach can be found in the data matrix attached as background material (see section below).

Conclusions:

L-TEE was tested for genetic toxicity in bacteria in accordance to OECD 471 (Ames test). Testing data on L-TEE shows no mutagenic activity in the Ames test (+/- metabolic activation). L-TME holds the same structure as L-TEE except that L-TME contains a methyl alkyl-side group to the ester bond and L-TEE an ethyl alkyl-side group. The toxicokinetic profile of L-TME and L-TEE is different in terms of the alcohol part (methanol and ethanol). Testing data (OECD 471, Supporting study, Klimisch score 2) retrieved from the REACH registration dossier of methanol (CAS No 67-56-1; EC No 200-659-6) shows that methanol is non-mutagenic in the Ames test (+/- S9).

Due to read across to negative mutagenicity data on L-TEE and further considering the negative mutagenicity data on methanol, L-TME is considered to be non-mutagenic.

Executive summary:

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 5000µg/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.

L-TME holds the same structure as L-TEE except that L-TME contains a methyl alkyl-side group to the ester bond and L-TEE an ethyl alkyl-side group. The toxicokinetic profile of L-TME and L-TEE is different in terms of the alcohol part (methanol and ethanol). Testing data (OECD 471, Supporting study, Klimisch score 2) retrieved from the REACH registration dossier of methanol (CAS No 67-56-1; EC No 200-659-6) shows that methanol is non-mutagenic in the Ames test (+/- S9).

Due to read across to negative mutagenicity data on L-TEE and further considering the negative mutagenicity data on methanol, L-TME is considered to be non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Additional information from genetic toxicity in vitro:

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 5000µg/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.

L-TME holds the same structure as L-TEE except that L-TME contains a methyl alkyl-side group to the ester bond and L-TEE an ethyl alkyl-side group. The toxicokinetic profile of L-TME and L-TEE is different in terms of the alcohol part (methanol and ethanol). Testing data (OECD 471, Supporting study, Klimisch score 2) retrieved from the REACH registration dossier of methanol (CAS No 67-56-1; EC No 200-659-6) shows that methanol is non-mutagenic in the Ames test (+/- S9).

Due to read across to negative mutagenicity data on L-TEE and further considering the negative mutagenicity data on methanol, L-TME is considered to be non-mutagenic.

Justification for selection of genetic toxicity endpoint

Key study

Justification for classification or non-classification

L-TEE was tested for mutation in accordance to OECD 471 (Ames test). Testing data on L-TEE shows no mutagenic activity in the Ames test (+/- metabolic activation). Due to the structural similarity to L-TEE (the L-Threonine part) and the differences in toxicokinetic properties (methanol and ethanol), the mutagenic activity of L-TME is described by the toxicity of methanol. However, methanol is shown non-mutagenic in Ames test. Based on read across to L-TEE taking into account differences in toxicokinetic properties, L-TME is considered to be non-mutagenic. Thus no classification for mutagenicity apply.