2-ethylhexyl 3-mercaptopropionate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

negative in OECD TG 471 and OECD TG 473;

based on read-across: negatve in OECD 476

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 14, 2022 - June 30, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

adopted 29th July, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Rat Liver S9 Fraction

Test concentrations with justification for top dose:

Experiment A with 3/20 h treatment/sampling time,
without S9 mix: 50, 100 and 150 μg/mL test item
with S9 mix: 500, 1000 and 2000 μg/mL test item

Experiment B with 20/20 h treatment/sampling time
without S9 mix: 50, 100, 125 and 150 μg/mL test item

Experiment B with 20/28 h treatment/sampling time
without S9 mix: 50, 100, 125 and 150 μg/mL test item

Experiment B with 3/28 h treatment/sampling time
with S9 mix: 500, 1000 and 2000 μg/mL test item

(selected on the basis of a pre-test on cytotoxicity)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 5E05 cells/dish
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
- Experiment A: 3-hour treatment, harvest 20 hours from the beginning of treatment
- Experiment B: 20-hour treatment, harvest 20 hours from the beginning of treatment / 20 (without S9 mix)- and 3-hour (with S9 mix) treatment, harvest 28 hours
from the beginning of treatment

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor: Colchicine (0.2 μg/mL) 2.5-3 hours prior to harvesting
- Methods of slide preparation and staining technique used including the stain used:
Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation becomes plasma free) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies.
300 well-spread metaphase cells containing 22 ± 2 chromosomes were scored per test item concentration as well as the negative and positive controls and were equally divided among the duplicates (150 metaphases/slide). Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.
Additionally, the number of polyploid and endoreduplicated cells were scored. The nomenclature and classification of chromosome aberrations were given based upon ISCN, 1985, and Savage, 1976, 1983.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- pretest for selection of concentrations: Relative Increase in Cell Counts (RICC)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Chromatid and chromosome type aberrations (gaps, deletions and exchanges), the number of polyploid and endoreduplicated cells

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.
Providing that all acceptability criteria are fulfilled, the test item is considered clearly negative if, in all experimental conditions examined:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test,
– all results are inside the distribution of the laboratory historical negative control data.

Both biological and statistical significance are considered together.
There is no requirement for verification of a clearly positive or negative response.

Statistics:

For statistical analysis the CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too. The data were checked for a linear trend in number of cells with aberrations (without gaps). with treatment dose using the adequate regression analysis by Microsoft
Excel software.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality.
Clear cytotoxicity in the range required by the guideline (55 ± 5 %) was observed at the highest concentrations without metabolic activation in Experiment A and experiment B (between 53 and 55%). In experiment A and in experiment B at the highest recommended concentration with metabolic activation 45% cytotoxicity was observed.

Conclusions:

In conclusion, Evabopol® 496D 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 presence of metabolic activation.
Thus, the test item EHMP is considered as being non-clastogenic in this system.

Executive summary:

The test item EHMP, dissolved in DMSO (Dimethyl sulfoxide), was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on cytotoxicity (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473:
Experiment A with 3/20 h treatment/sampling time
without S9 mix: 50, 100 and 150 μg/mL test item
with S9 mix: 500, 1000 and 2000 μg/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 50, 100, 125 and 150 μg/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 50, 100, 125 and 150 μg/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 500, 1000 and 2000 μg/mL test item
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).
No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item.
Clear cytotoxicity in the range required by the guideline (55 ± 5 %) was observed at the highest concentrations without metabolic activation in Experiment A and experiment B (between 53 and 55%). In experiment A and in experiment B at the highest recommended concentration with
metabolic activation 45% cytotoxicity was observed.
In experiment A, 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.
In experiment B no increases in cells carrying structural chromosomal aberrations compared to concurrent controls or in comparison with the range of historical controls were observed in the absence (20-hour treatment/28-hour sampling time) 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.

The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.
In conclusion, EHMP 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 presence of metabolic activation.
Thus, the test item is considered as being non-clastogenic in this system.

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:

There are no relevant variations in qualitative properties among source substances and the same potency is predicted for all target substances. This is Scenario 4 of the RAAF1. Substances MMP, BuMP, EHMP, iOMP, iC13MP, and ODMP are different alkyl esters of a common acid, 3-mercaptopropionic acid (3-MPA).
This scenario covers the category approach for which the read-across hypothesis is based on the assumption, that toxicity of compounds in this category are driven by a common toxophore. This approach serves to use existing data on genotoxicity, acute toxicity, repeated-dose toxicity and reproductive toxicity endpoints for substances in this category. 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 differences in strength of the effects forming a regular pattern. This corresponds to Scenario 4 of the RAAF. The substances MMP, BuMP, EHMP, iOMP, iC13MP, and ODMP are esters of a common acid, 3-mercaptopropionic acid (3-MPA). All category members share the same mercaptopropionic acid moiety with one free SH group per MPA unit. The MPA unit with free SH is a prerequisite for this category.
The observed differences in effect levels (higher effect levels with increasing carbon chain length were observed in the available acute oral toxicity studies) are assumed to be mainly due to differences in molecular weight (corrections will be made for these differences) and decreasing bioavailability with increasing carbon chain length (no corrections are made for this effect; a worst-case approach is applied here, since based on the available data no exact quantification for bioavailability differences is possible at the moment).
It can be predicted with high confidence that the substances within this category will lead to the same type of effects. The main driver for toxicity is the free SH group of the MPA moiety.
Beside structural similarities and the common toxophore, the MPA moiety, category members are also likely to have similar metabolites. As explained in the Toxicokinetics section, substances are predicted to be rapidly hydrolysed into 3-MPA and the respective alcohol after absorption. According to low toxicity of the corresponding alcohols (Table 8), this would support the role of the MPA moiety as toxophore, as well as propose scenario 3 of the RAAF as applicable for this category approach. However, no experimental toxicokinetic data to support this hypothesis are available by now. To proof this hypothesis, simulated gastric acid hydrolysis studies, as well as in-vitro metabolism studies using liver microsomes will be conducted. Based on the results of these studies, scenario selection will be revaluated.

For detailed information refer to section 13.2.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

The category members 3-MPA, iOMP and MMP showed negative results in gene mutation assays in mammalian cells. Based on the category approach, EHMP is expected to show similar effects. Therefors, BuMP is considered not to induce gene mutation in mammalian cell.

As Toxikokinetic literature data indicate that after absorption rapid ester hydrolysis accurs to all category members, toxicokinetic in vitro studies are planned to gain better insight on toxicokinetic properties of the substances. Depending on the results of metabolism and simulated gastric acid hydrolysis studies, scenario selection will be revaluated.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 21, 2022 - July 01, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21st July 1997, corrected 26th June 2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

dated May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his (S. typhimurium) / trp (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Rat Liver S9 Fraction

Test concentrations with justification for top dose:

5000, 1600, 500, 160, 50 and 16 μg/plate (tested up to limit dose)

Vehicle / solvent:

DMSO; Based on the results of the preliminary experiments (solubility and concentration range finding tests) dimethyl sulfoxide (DMSO) was found as appropriate vehicle for preparing the test item solutions.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine (TA98 without S9)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicates
- Number of independent experiments: 2; initial mutation test (plate incorporation test) and a confirmatory mutation test (pre-incubation test)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min at 37ºC
- Exposure duration/duration of treatment: 48 h


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs

Evaluation criteria:

A result is considered positive if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2
uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9).

In the initial and confirmatory mutation tests, unequivocal inhibitory effect of the test item on bacterial growth was observed.

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations in the genome of the strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and of Escherichia coli WP2 uvrA.
In conclusion, the test item EHMP has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item EHMP was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.
Bacterial strains, exogenous metabolic activation:
Salmonella typhimurium TA98, target mutation: hisD3052; mutation type: frameshift;
Salmonella typhimurium TA100, target mutation: hisG46; mutation type: base pair substitution;
Salmonella typhimurium TA1535, target mutation: hisG46; mutation type: base pair substitution;
Salmonella typhimurium TA1537, target mutation: hisC3076; mutation type: frameshift;
Escherichia coli WP2 uvrA, target mutation: trpE; mutation type: base pair substitution.
Exogenous metabolic activation:
The experiments were carried out in the presence and absence of a cofactor supplemented post-mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats. The S9 mix contained 10 % (v/v) S9.Experimental phases:
The study included preliminary solubility test, a preliminary concentration range finding test (informatory toxicity test applying the plate incorporation method), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test).

Vehicle, test item concentrations, rationale for dose selection:
Based on the results of the solubility test and the concentration range finding test the test item was dissolved in dimethyl sulfoxide (DMSO) and the following concentrations of the test item were prepared and investigated in the main experiments: ±S9: 5000, 1600, 500, 160, 50 and 16 μg/plate.
For the selected concentration range, the noticed cytotoxicity (noticed in the informatory toxicity test), the solubility (adequately soluble in the applied test system), and the laboratory’s experience with used strains (for concentration choice at TA1535, TA1537 and Escherichia coli WP2 uvrA strains) were taken into consideration based on the recommendations in OECD 471 guideline.
At the preparation of the test item solutions any correction (multiplier) factor was not taken into consideration, the concentrations were based on the final formulation as is.

Validity of the Study:
In the performed experiments all of the validity criteria, regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analysable concentration levels were fulfilled.
The revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO)) plates with and without S9 Mix demonstrated the characteristic mean numbers of spontaneous revertants that were mostly in line with the corresponding historical control data ranges. The slightly outlier value in the initial mutation test case of Escherichia coli WP2 uvrA strain (−S9) was considered acceptable, remained within the biological variability range (no extreme outlier).

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases.

Solubility, precipitation:
No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9).

Cytotoxicity results:
In the initial and confirmatory mutation tests, unequivocal inhibitory effect of the test item on bacterial growth was observed. The inhibitory effect was indicated by affected background lawn development: reduced or slightly reduced background lawn and/or decreased revertant colony counts (absent revertants or revertants below the actual vehicle control ranges and/or corresponding historical control data ranges).
In general, 500 μg/plate, noticed following the plate incorporation procedure (initial mutation test) in Salmonella typhimurium TA100 in the absence and presence of exogenous metabolic activation (±S9) and following the pre-incubation procedure (confirmatory mutation test) in all Salmonella typhimurium strains, in absence (−S9), in Salmonella typhimurium TA100 also in the presence of exogenous metabolic activation (+S9) was
considered as the lowest concentration showing unequivocal cytotoxicity.

Mutagenicity results:
No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with Evabopol® 496D at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

Conclusion:
The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations in the genome of the strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and of Escherichia coli WP2 uvrA.
In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The substances MMP, BuMP, EHMP, iOMP, iC13MP, and ODMP are esters of a common acid, 3-mercaptopropionic acid (3-MPA). All category members share the same mercaptopropionic acid moiety with one 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 MPA, MMP, BuMP, EHMP, iOMP, and ODMP.

 

3-MPA

The mutagenic potential of 3-MPA was investigated in a bacterial reverse mutation assay (plate incorporation method) using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors) in accordance with OECD TG 471. The test material was  tested up to the maximum recommended dose level of 5000 pg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
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. 3-MPA was considered to be non-mutagenic under the conditions of this test.

 

BuMP

The test item BuMP was tested for mutagenic activity using the Bacterial Reverse Mutation Assay (plate incorporation test and confirmatory pre-incubation test) using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and Escherichia coli WP2 uvrA, with and without metabolic activation in accordance with OECD TG 471. The test item was tested up to cytotoxic concentrations.

No biologically relevant increases in revertant colony numbers were observed for any of the five test strains at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. In conclusion, BuMP has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

 

EHMP
The test item EHMP was tested for mutagenic activity using the Bacterial Reverse Mutation Assay (plate incorporation test and confirmatory pre-incubation test) using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and Escherichia coli WP2 uvrA, with and without metabolic activation in accordance with OECD TG 471. The test item was tested up to cytotoxic concentrations.

No biologically relevant increases in revertant colony numbers were observed for any of the five test strains at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. In conclusion, EHMP has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

 

iOMP

The test item iOMP was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA with and without metabolic activation in accordance with OECD TG 471. The test item was tested up to cytotoxic concentrations.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed at any concentration level, neither in the presence nor absence of metabolic activation. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, iOMP is considered to be non-mutagenic in this bacterial reverse mutation assay.

 

ODMP

The test item ODMP was tested for mutagenic activity using the Bacterial Reverse Mutation Assay (plate incorporation test and confirmatory pre-incubation test) using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and Escherichia coli WP2 uvrA, with and without metabolic activation in accordance with OECD TG 471. No cytotoxicity was observed, the test item was tested up to the limit concentration of 5000 μg/plate

No biologically relevant increases in revertant colony numbers were observed for any of the five test strains at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. In conclusion, ODMP has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

 

 

Chromosome aberration assay

Negative chromosomal aberration assays are available for MPA and MMP; An in-vitro chromosomal aberration study in mammalian cells is planned with EHMP to cover the range of alkyl chain length in this category and strengthen the hypothesis, that the esters do not lead to chromosomal aberration.

 

3-MPA

The clastogenic potential of 3-MPA was investigated in a chromosome aberration assay (human lymphocytes; with and without metabolic activation) according to OECD TG 473.

The test material was moderately toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a concentration range that included a concentration level that induced approximately 50% mitotic inhibition.

The test material was considered to be non-clastogenic to human lymphocytes in vitro.

 

MMP

MMP did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) with and without metabolic activation when tested up to cytotoxic concentrations according to OECD TG 473.

 

EHMP

The test item EHMP was tested in a chromosome aberration assay in V79 cells with and without metabolic activation in accordance with OECD TG 473. EHMP 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 presence of metabolic activation.
Thus, the test item is considered as being non-clastogenic in this system.

 

Gene mutation assay in mammalian cells

Negative mouse lymphoma assays are available for MPA, MMP, and iOMP.

 

3-MPA

The mutagenic potential of 3-MPA was investigated in a mouse lymphoma assay according to OECD TG 476 (TK assay). L5178Y TK +/- 3.7.2¢ mouse lymphoma cells were treated with the test material at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation or in a second experiment 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment, using a dose range that included the 10 mM limit dose in the 4-hour exposure groups and also a dose level that induced the optimum level of toxicity in the 24-hour exposure group. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

 

MMP

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

MMP did not induce a dose-dependent increase in mutation frequency of two or more test concentrations by at least two to three-fold higher than the solvent control. MMP is not considered to be mutagenic in this test system.

 

iOMP

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

Growth inhibition was observed in the main experiment with and without metabolic activation.
In the main experiment with and without metabolic activation no biologically relevant increase of mutants was found after treatment with the test. No dose-response relationship was observed.
In addition, colony sizing did not indicate potential 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 is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line LS178Y.

 

Conclusion

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