2-ethyl-2-[(3-mercapto-1-oxopropoxy)methyl]propane-1,3-diyl bis[3-mercaptopropionate]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

Currently, do data on toxicokinetics/metabolism is available for this category. Based on structural features (e.g. sterical hindrance) it is however assumed, that ester cleavage would not be fast and complete, especially since the substances contain up to 6 ester functions, which are in addition sterically shielded. Therefore, it seems more reasonable to base the category hypothesis on structural similarity.
In addition, it is not clear yet, whether the strength of the effects vary in a predictable manner, or if no relevant variations occur. However, there are variations in structure (number of ester bonds and consequently number of free -SH groups) and physicochemical properties (especially water solubility and log Kow). It is assumed that these variations will also be reflected by variations in effect levels. Therefore, scenario 4 is the working hypothesis for the time being.
More data points within the category are needed to further strengthen the category hypothesis. The scenario selection will be re-evaluated after the studies are finished.
This currently selected scenario covers the category approach for which the read-across hypothesis is based on structural similarity. For the REACH information requirement under consideration, the property investigated in studies conducted with different source substances is used to predict the property that would be observed in a study with the target substance if it were to be conducted. Similar properties are observed for the different source substances; this may include absence of effects for every member of the category.
There are expected to be differences in strength of the effects forming a regular pattern. The prediction will be based on a worst-case approach. The read-across is a category approach based on the hypothesis that the substances in this category share structural similarities with common functional groups. This approach serves to use existing data on acute toxicity, repeated-dose toxicity, and reproductive toxicity endpoints for substances in this category.
The hypothesis corresponds to Scenario 4 of the RAAF. The substances GDMP, TMPMP, PETMP, and Di-PETMP are esters of a common acid, 3-mercaptopropionic acid (3-MPA). The key functionality of the substances within this category is the presence of free SH-groups. It is hypothesised that the strength of effects correlates with the number of SH-groups. In addition, differences in bioavailability are expected to influence the strength of effects.
For details, please refer to the category document attached to Iuclid section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1998

Deviations:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Metabolic activation:

with and without

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: established vehicle for substances with low water solubility

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 7.8/190 µg/mL, -/+ S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at concentrations of 250 µg/mL and higher.

RANGE-FINDING/SCREENING STUDIES:
According to the used guidelines the highest recommended concentration is 5000 µg/mL. The test item was dissolved in DMSO and diluted in cell
culture medium. The highest concentration evaluated in the preexperiment was 5000 µg/mL. The relative mitotic index was used as parameter for toxicity. The concentrations evaluated in the main experiment based on the results obtained in the pre-experiment-

COMPARISON WITH HISTORICAL CONTROL DATA:
compliant

Conclusions:

Based on read-across from PETMP and GDMP, TMPMP does not induce chromosomal aberrations in mammalian cells.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

Currently, do data on toxicokinetics/metabolism is available for this category. Based on structural features (e.g. sterical hindrance) it is however assumed, that ester cleavage would not be fast and complete, especially since the substances contain up to 6 ester functions, which are in addition sterically shielded. Therefore, it seems more reasonable to base the category hypothesis on structural similarity.
In addition, it is not clear yet, whether the strength of the effects vary in a predictable manner, or if no relevant variations occur. However, there are variations in structure (number of ester bonds and consequently number of free -SH groups) and physicochemical properties (especially water solubility and log Kow). It is assumed that these variations will also be reflected by variations in effect levels. Therefore, scenario 4 is the working hypothesis for the time being.
More data points within the category are needed to further strengthen the category hypothesis. The scenario selection will be re-evaluated after the studies are finished.
This currently selected scenario covers the category approach for which the read-across hypothesis is based on structural similarity. For the REACH information requirement under consideration, the property investigated in studies conducted with different source substances is used to predict the property that would be observed in a study with the target substance if it were to be conducted. Similar properties are observed for the different source substances; this may include absence of effects for every member of the category.
There are expected to be differences in strength of the effects forming a regular pattern. The prediction will be based on a worst-case approach. The read-across is a category approach based on the hypothesis that the substances in this category share structural similarities with common functional groups. This approach serves to use existing data on acute toxicity, repeated-dose toxicity, and reproductive toxicity endpoints for substances in this category.
The hypothesis corresponds to Scenario 4 of the RAAF. The substances GDMP, TMPMP, PETMP, and Di-PETMP are esters of a common acid, 3-mercaptopropionic acid (3-MPA). The key functionality of the substances within this category is the presence of free SH-groups. It is hypothesised that the strength of effects correlates with the number of SH-groups. In addition, differences in bioavailability are expected to influence the strength of effects.
For details, please refer to the category document attached to Iuclid section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase

Metabolic activation:

with and without

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: established vehicle for substances with low water solubility

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at >= 40 µg/mL (-S9); >=313 µg/mL (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on read across from PETMP, TMPMP is predicted to be negative in the mouse lymphoma assay, with and without metabolic activation

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

Currently, do data on toxicokinetics/metabolism is available for this category. Based on structural features (e.g. sterical hindrance) it is however assumed, that ester cleavage would not be fast and complete, especially since the substances contain up to 6 ester functions, which are in addition sterically shielded. Therefore, it seems more reasonable to base the category hypothesis on structural similarity.
In addition, it is not clear yet, whether the strength of the effects vary in a predictable manner, or if no relevant variations occur. However, there are variations in structure (number of ester bonds and consequently number of free -SH groups) and physicochemical properties (especially water solubility and log Kow). It is assumed that these variations will also be reflected by variations in effect levels. Therefore, scenario 4 is the working hypothesis for the time being.
More data points within the category are needed to further strengthen the category hypothesis. The scenario selection will be re-evaluated after the studies are finished.
This currently selected scenario covers the category approach for which the read-across hypothesis is based on structural similarity. For the REACH information requirement under consideration, the property investigated in studies conducted with different source substances is used to predict the property that would be observed in a study with the target substance if it were to be conducted. Similar properties are observed for the different source substances; this may include absence of effects for every member of the category.
There are expected to be differences in strength of the effects forming a regular pattern. The prediction will be based on a worst-case approach. The read-across is a category approach based on the hypothesis that the substances in this category share structural similarities with common functional groups. This approach serves to use existing data on acute toxicity, repeated-dose toxicity, and reproductive toxicity endpoints for substances in this category.
The hypothesis corresponds to Scenario 4 of the RAAF. The substances GDMP, TMPMP, PETMP, and Di-PETMP are esters of a common acid, 3-mercaptopropionic acid (3-MPA). The key functionality of the substances within this category is the presence of free SH-groups. It is hypothesised that the strength of effects correlates with the number of SH-groups. In addition, differences in bioavailability are expected to influence the strength of effects.
For details, please refer to the category document attached to Iuclid section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no 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:

no 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:

no 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

GDMP and PETMP showed a no signs of genotoxicity in an bacterial reverse mutation assay. Based on the category approach TMPMP is considered to be non genotoxic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The substances GDMP, TMPMP, PETMP, and Di-PETMP are esters of a common acid, 3-mercaptopropionic acid (3-MPA). All category members share the same mercaptopropionic acid moiety with two to 6 free SH group per MPA unit. The MPA unit with free SH is a prerequisite for this category. A justification for read-across is attached to Iuclid section 13.

 

Bacterial reverse mutation assay

Negative Ames tests are available for GDMP and PETMP. 

 

GDMP was examined for iits mutagenic potential in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 and Escherichia coli WP2 uvr A in two independent experiments, each carried out without and with metabolic activation. The first experiment was carried out as a plate incorporation test and the second as a preincubation test.
Cytotoxicity was noted at the top concentration of 5000 μg GDMP/plate. No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for GDMP, tested up to a cytotoxic concentration of 5000 μg/plate, in the Salmonella typhimurium and in the Escherichia coli test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

 

The genotoxic potential of the test item PETMP was assessed in a Bacterial reverse mutation assay according to OECD Guideline 471 and EU method B13/14. Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with solutions of the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A white, cloudy precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

The test material was considered to be non-mutagenic under the conditions of this test.

 

Chromosome aberration assay

Negative chromosomal aberration assays are available for GDMP and PETMP.

 

The test item GDMP, dissolved in DMSO (Dimethyl sulfoxide), was tested in a chromosome aberration assay in V79 cells in two independent experiments in accordance with the current OECD Guideline 473. For the cytogenetic experiments the concentrations were selected on the basis of a pre-test on cytotoxicity (without and with metabolic activation).
Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture).
Clear cytotoxicity in the range required by the guideline (55 ± 5 %) was observed at the highest concentrations with and without metabolic activation in both experiments.
In both experiments, no increases in cells carrying structural chromosomal aberrations compared to concurrent controls or in comparison with the range of historical controls were observed, neither in the absence nor in the presence of metabolic activation. All values for aberrant cells were within the historical control range of 2-5 aberrant cells excluding gaps.
There were no polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.
In conclusion, GDMP did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to the cytotoxic concentrations in the absence and up to the maximum recommended concentration in the presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.

 

To investigate the potential of PETMP to induce structural chromosome aberrations in Chinese hamster V79 cells in the presence and absence of metabolic activation, an in vitro chromosome aberration assay according to OECD TG  473 was carried out. PETMP was tested up to cytotoxic concentrations.

The chromosomes were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations.

In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all concentrations treated with the test item were within the historical control data of the negative control.

In experiment I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls.

In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item PETMP did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.

Therefore, the test item is considered to be non-clastogenic.

 

Mammalian cell gene mutation assay

A negative mouse lymphoma assay is available for PETMP.

 

The test item PETMP was assessed for its potential to induce mutations at the thymidine kinase locus using the mouse lymphoma cell line L5178Y with and without metabolic activation in accordance with OECD Guideline 476.

No biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.

Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item PETMP is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cellline L5178Y when tested up to cytotoxic concentrations.

 

 

Conclusion

The entirety of available genotoxicity data in combination with the absence of structural alerts for genotoxicity in this category will be used to strengthen the weight of the evidence for non-genotoxicity.