2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-7-oxo-8-oxa-3,5-dithia-4-phosphatetradecanoate 4-oxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria (Ames test)

In vitro gene mutation study in mammalian cells (mouse lymphoma) - read across

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

The tests were carried out in accordance with the method published by AMES et al., and with the modifications described
by the Ministry of Labour of Japan.

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

TEST MATERIAL (as stated in study report): TK 12 184; common name D 16-51

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor / Batch No. Mixt. 4/5/6

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of liver from rats induced with Aroclor 1254

Test concentrations with justification for top dose:

5, 10, 50, 100, 500, 1000 and 5000 µg/0.1 ml. The top dose is the highest dose recommeneded by test guidelines.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene

Details on test system and experimental conditions:

Each Petri dish contained: 1) approx. 20 ml of minimum agar (Agar, Difco Laboratories, Detroit, Michigan, U.S.A., plus salts (Vogel-Bonner Medium E) and glucose), 2) 0.1 ml of the solution of the test substance or the vehicle and 0.1 ml of a bacterial culture (in nutrient broth, Difco Laboratories, Detroit, Michigan, U.S.A., 0.8% plus 0.5% NaCl) in 2.0 ml of soft agar. The soft agar was composed of: 100 ml of 0.6% agar solution with 0.6% NaCl and 10 ml of a solution of 1-histidine, 0.5 mM (Fluka, Buchs, Switzerland) and +biotin 0.5 mM (Fluka, Buchs, Switzerland). In the experiments in which the substance was metabolically activated, 0.5 ml of an activation mixture was added also.

In the experiments without and with the addition of microsomal activation mixture three Petri dishes were prepared per strain
and per group (i.e. per concentration or per control group).

The plates were incubated for about 48 hours at 37 C in darkness.

Rationale for test conditions:

per referenced methods

Evaluation criteria:

The test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration.

Statistics:

Arithmetic means were calculated for each test group.

Species / strain:

other: TA98, TA100, TA1538, TA1535, TA1537, WP2uvra

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of TK 12 184 revealed no marked deviations.

Conclusions:

Under these standard test conditions, there was no evidence of the induction of point mutations by TK 12 184 in S. typhimurium or E. coli at concentrations up to 5000 µg/plate with and without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

- Supplier: Arkema France
- Name on the study plan : 2-ETHYLHEXYL THIOGLYCOLATE
- Synonym: 2-EHTG
- Batch number: 12.11.07.G
- Description: colourless liquid
- Storage conditions: at room temperature
- Purity: 99.77% (w/w) (analysis of 21 January 2008)
99.88% (w/w) (analysis of 06 August 2009)

Target gene:

TK (Thymidine Kinase) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y cells, were obtained from ATCC (American Type Culture Collection, Manassas, USA),
by the intermediate of Biovalley (Marne-La-Vallée, France).

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from the liver of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

- Experiments without S9 mix:
· 0.025, 0.05, 0.1, 0.2, 0.4, 0.6 and 0.8 mM for the first experiment (3-hour treatment),
· 0.010, 0.020, 0.041, 0.081, 0.16, 0.32 and 0.49 mM for the second experiment (24-hour treatment).
- Experiments with S9 mix:
· 0.013, 0.025, 0.05, 0.1, 0.2, 0.4 and 0.6 mM for the first experiment,
· 0.05, 0.1, 0.2, 0.4, 0.6, 0.8 and 1 mM for the second experiment,
· 0.125, 0.25, 0.5, 0.75, 1, 1.5, 2, 3 and 4 mM for the third experiment,
· 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10 mM for the fourth experiment.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9 mix: methylmethane sulfonate (MMS), 25 μg/mL, (3-hour treatment) or 5 μg/mL (24-hour treatment), With S9 mix: Cyclophosphamide (CPA), 3 μg/mL.

Details on test system and experimental conditions:

After a preliminary toxicity test, 2-ETHYLHEXYL THIOGLYCOLATE was tested for mutagenicity in independent experiments, with and/or without a metabolic activation system (S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254).

Two experiments were performed without S9 mix (3- and 24-hour treatments) and four experiments with S9 mix (3-hour treatment).

Approximately 0.5 x 106 (3-hour treatment) or 0.15 x 106 (24-hour treatment) cells/mL in 20 mL culture medium with 5% horse serum were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%), at 37°C. For the 24-hour treatment, the incubation at 37°C was performed with a gentle shaking.

Cytotoxicity was measured by assessment of adjusted relative total growth (Adj. RTG) and relative suspension growth (Adj. RSG) as well as cloning efficiency following the expression time (CE2).

The number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype.

Evaluation criteria:

IWGT recommendations were followed for the determination of a positive result which should
fulfill the following criteria:
· at least at one concentration the mutation frequency minus the mutation frequency of the vehicle control equals or exceeds the global evaluation factor (126 x 10-6 for the microtiter method),
· and a dose-related trend is demonstrated by a statistically significant trend test.

Unless considered as clearly positive, the reproducibility of a positive effect should be confirmed. Noteworthy increases in the mutation frequency observed only at high levels of cytotoxicity (RTG lower than 10%), but with no evidence of mutagenicity at concentrations with RTG between 10 and 20%, is considered as positive result.

A test item may be determined to be non-mutagenic when there is no culture showing an Adj. RTG value between 10-20% if (e):
· there is at least one negative data point between 20 and 25% Adj. RTG and no evidence on mutagenicity in a series of data points between 100 to 20% Adj. RTG,
· there is no evidence of mutagenicity in a series of data points between 100 to 25% and there is also a negative data point between 10 and 1% Adj. RTG.

Statistics:

Trend test

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The cloning efficiencies CE2 and the mutation frequencies of the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered as valid.

Since the test item was toxic in the preliminary test, the choice of the highest concentration retained for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in Adj. RTG).

- Experiments without S9 mix
. Cytotoxicity
Following the 3-hour treatment (Tables 2 and 3), a slight to marked toxicity was noted at concentrations = 0.1 mM, as shown by a 24-69% decrease in Adj. RTG.
Following the 24-hour treatment (Tables 4 and 5), a marked to severe toxicity was induced at concentrations = 0.16 mM, as shown by a 67 to 100% decrease in Adj. RTG.
. Mutagenicity
Following the 3-hour treatment (Table 3), no noteworthy increase in the mutation frequency was observed at concentrations up to 0.8 mM, which showed a 69% decrease in Adj. RTG.
Following the 24-hour treatment (Table 5), a slight increase in the mutation frequency was observed at 0.32 mM. This increase (+125 x 10-6 compared to the vehicle control mean value of 137 x 10-6) did not reach the global evaluation factor of +126 x 10-6 and was only observed at a high level of cytotoxicity (Adj. RTG lower than 10%). Therefore, it was not considered as biologically relevant.

- Experiments with S9 mix
At the end of the 3-hour treatment, a slight to moderate emulsion was noted in the culture medium at concentrations = 2.5 mM in the fourth experiment.
. Cytotoxicity
In the first experiment (Tables 6 and 7), a slight to moderate toxicity was induced at concentrations = 0.2 mM, as shown by a 29 to 50% decrease in Adj. RTG.
In the second experiment (Tables 8 and 9), a slight to moderate toxicity was induced at concentrations = 0.4 mM, as shown by a 24 to 49% decrease in Adj. RTG.
In the third experiment (Tables 10 and 11), no toxicity was induced at any of the concentrations tested.
In the fourth experiment (Tables 12 and 13), a slight to marked toxicity was induced at concentrations = 0.313 mM, as shown by a 25 to 74% decrease in Adj. RTG.
. Mutagenicity
In the two first main experiments (Tables 7 and 9), no noteworthy increase in the mutation frequency was induced at concentrations up to 1 mM, which showed a maximum of 50% decrease in Adj. RTG. Since the concentrations tested did not reach a toxicity level of 80-90% decrease in Adj. RTG, a third additional experiment was undertaken under the same experimental conditions but using a higher range of concentrations (Table 11). In this experiment, no noteworthy increase in the mutation frequency was induced at concentrations up to 4 mM but the required toxicity level was still not reached. Therefore, a fourth additional experiment was undertaken using an even higher range of concentrations (up to 10 mM, Table 13). In these conditions, the top concentration of 10 mM induced a 74 % decrease in Adj. RTG and no noteworthy increase in the mutation frequency was observed at any of the tested concentrations.

Conclusions:

Although 2-ETHYLHEXYL THIOGLYCOLATE caused inconsistent cytotoxic effects and the requested level of 10-20% Adj.RTG was not reached in all experiments, the study clearly shows that 2-ETHYLHEXYL THIOGLYCOLATE does not have a mutagenic potential in the mouse lymphoma assay, either with or without a metabolic activation system.

Executive summary:

2-Ethylhexyl thioglycolate was tested in an in vitro gene mutation assay using mammalian cells cultures both in the absence and presence of metabolic activation (S9 mix), according to the OECD No. 476 Guideline and in compliance with the Principles of Good Laboratory Practice. Mouse lymphoma L5178Y (TK+/-) cells cultured in vitro were exposed to 2-Ethylhexyl thioglycolate in DMSO for 3 or 24 hours at concentrations up to 0.8 mM in absence of metabolic activation and for 3 hours at concentrations up to 10 mM in presence of metabolic activation. Appropriate positive controls were used and showed a statistical increase in mutant colonies. After a 48 rest period, cells were then incubated for mutagenicity evaluation with trifluorothymidine.

Although 2-ethylhexyl thioglycolate caused inconsistent cytotoxic effects and the requested level of 10-20% Adj.RTG was not reached in all experiments, the study clearly shows that 2-ethylhexyl thioglycolate does not have a mutagenic potential in the mouse lymphoma assay, either with or without a metabolic activation system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]
The read across substance, 2-ethylhexyl mercaptoacetate, is a presursor and impurity in the commercial form of the registered substance (product name D 16-051). More importantly, 2-ethyl hexyl mercaptoacetate is also an expected hydrolysis degradation product of the registered substance.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]
The commercial form of the registered substance (product name D 16-051), contains approximately 4% 2-ethylhexyl mercaptoacetate.

3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]
In addition to being a precursor and impurity in the commercial form of the registered substance, 2-ethylhexyl mercaptoacetate is an expected hydrolysis degradation product of the registered substance. It is therefore considered to be a relevant surrogate substance.

4. DATA MATRIX
The registered substance was negative in the Ames test at concentrations up to 5000 µg/plate with and without metabolic activation, and did not produce cytogenetic changes in the bone marrow of Chinese hamster treated by oral gavage up to a limit dose of 3,000 mg/kg.

The read across substance, 2-ethylhexyl mercaptoacetate was negative in the in vitro gene mutations assays on bacteria (Ames test) and mammalian cells (mouse lymphoma assay), both in the presence and absence of metabolic activation, and was negative in the in vivo mouse micronucleus test.

Reason / purpose for cross-reference:

read-across source

Specific details on test material used for the study:

- Supplier: Arkema France
- Name on the study plan : 2-ETHYLHEXYL THIOGLYCOLATE
- Synonym: 2-EHTG
- Batch number: 12.11.07.G
- Description: colourless liquid
- Storage conditions: at room temperature
- Purity: 99.77% (w/w) (analysis of 21 January 2008)
99.88% (w/w) (analysis of 06 August 2009)

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The cloning efficiencies CE2 and the mutation frequencies of the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered as valid.

Since the test item was toxic in the preliminary test, the choice of the highest concentration retained for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in Adj. RTG).

- Experiments without S9 mix
. Cytotoxicity
Following the 3-hour treatment (Tables 2 and 3), a slight to marked toxicity was noted at concentrations = 0.1 mM, as shown by a 24-69% decrease in Adj. RTG.
Following the 24-hour treatment (Tables 4 and 5), a marked to severe toxicity was induced at concentrations = 0.16 mM, as shown by a 67 to 100% decrease in Adj. RTG.
. Mutagenicity
Following the 3-hour treatment (Table 3), no noteworthy increase in the mutation frequency was observed at concentrations up to 0.8 mM, which showed a 69% decrease in Adj. RTG.
Following the 24-hour treatment (Table 5), a slight increase in the mutation frequency was observed at 0.32 mM. This increase (+125 x 10-6 compared to the vehicle control mean value of 137 x 10-6) did not reach the global evaluation factor of +126 x 10-6 and was only observed at a high level of cytotoxicity (Adj. RTG lower than 10%). Therefore, it was not considered as biologically relevant.

- Experiments with S9 mix
At the end of the 3-hour treatment, a slight to moderate emulsion was noted in the culture medium at concentrations = 2.5 mM in the fourth experiment.
. Cytotoxicity
In the first experiment (Tables 6 and 7), a slight to moderate toxicity was induced at concentrations = 0.2 mM, as shown by a 29 to 50% decrease in Adj. RTG.
In the second experiment (Tables 8 and 9), a slight to moderate toxicity was induced at concentrations = 0.4 mM, as shown by a 24 to 49% decrease in Adj. RTG.
In the third experiment (Tables 10 and 11), no toxicity was induced at any of the concentrations tested.
In the fourth experiment (Tables 12 and 13), a slight to marked toxicity was induced at concentrations = 0.313 mM, as shown by a 25 to 74% decrease in Adj. RTG.
. Mutagenicity
In the two first main experiments (Tables 7 and 9), no noteworthy increase in the mutation frequency was induced at concentrations up to 1 mM, which showed a maximum of 50% decrease in Adj. RTG. Since the concentrations tested did not reach a toxicity level of 80-90% decrease in Adj. RTG, a third additional experiment was undertaken under the same experimental conditions but using a higher range of concentrations (Table 11). In this experiment, no noteworthy increase in the mutation frequency was induced at concentrations up to 4 mM but the required toxicity level was still not reached. Therefore, a fourth additional experiment was undertaken using an even higher range of concentrations (up to 10 mM, Table 13). In these conditions, the top concentration of 10 mM induced a 74 % decrease in Adj. RTG and no noteworthy increase in the mutation frequency was observed at any of the tested concentrations.

Conclusions:

The surrogate substance 2-ethylhexyl mercaptoacetate (EC No. 231 -626 -4), which is an impurity in D16-051 and also an expected hydrolysis degradation product of D 16-051, was negative in a guideline (OECD 476) mouse lymphoma assay with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo cytogenetics in hamster bone marrow

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 84-2

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

TEST MATERIAL (as stated in study report): TK 12 184

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor / Batch No. Mixt. 4/5/6

Species:

hamster, Chinese

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: males - 23 - 32 g; females - 20 - 28 g
- Housing: individual
- Diet (e.g. ad libitum): Standard diet: NAFAG No.924
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23 deg C
- Humidity (%): 60-80%
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

distilled water

Details on exposure:

dosage volume of 20 mL/kg

Duration of treatment / exposure:

2 consecutive days

Frequency of treatment:

once per day

Post exposure period:

24 h

Dose / conc.:

750 mg/kg bw/day (actual dose received)

Dose / conc.:

1 500 mg/kg bw/day (actual dose received)

Dose / conc.:

3 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

negative and positive controls: 3
treated groups: 6

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide - 128 mg/kg in 20 mL/kg of distilled water

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

Bone marrow was harvested from the shafts of both femurs. In a siliconized pipette filled with approx. 0.5 µL rat serum the bone marrow was drawn up. In order to receive a homogeneous suspension the content of pipette was aspirated gently about three times. Small drops of the mixture were transferred on the end of a slide, spread out by pulling it behind a polished cover glass and the preparations were air-dried. Three hours later, the slides were stained in undiluted May-Grünwald solution for 2 min then in May-Grünwald solution/water 1/1 for 2 min and then in Giemsa's, 40% for 20 min. After being rinsed in methanol 55% for 5-8 sec and washed off twice in water, they were left immersed in water for approx, 2 min. After rinsing with distilled water and.air-drying, the slides were cleared in Xylol and mounted in Eukitt.

Evaluation criteria:

The slides of three female and three male animals each of the negative control group, the positive control group and of the groups treated with various doses of TK 12 184 were examined. 1000 bone marrow interphase cells each were scored per animal and the following anomalies were registered: a) Single Jolly bodies, b) fragments of nuclei in erythrocytes, c) micronuclei in erythroblasts, d) micronuclei in leucopoietic cells, e) polyploid cells.

Statistics:

The significance of difference was assessed by chi square test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The bone marrow smears from animals treated with various doses of TK 12 184 showed no significant difference from the control. The incidence.of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group.

By contrast, a "positive control" experiment with cyclophosphamide (128 mg/kg) yielded 13.8% cells with anomalies of nuclei. This is significantly different from the controls treated with the vehicle (distilled water) alone.

Conclusions:

Under these test conditions no evidence of mutagenic effects was obtained in Chinese hamsters treated with TK 12 184 by oral gavage up to a limit dose of 3,000 mg/kg.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

TK 12 184 was negative in the Ames test at concentrations up to 5000 µg/plate with and without metabolic activation, and did not produce cytogenetic changes in the bone marrow of Chinese hamster treated by oral gavage up to a limit dose of 3,000 mg/kg.

An impurity in TGEPO, 2-ethylhexyl mercaptoacetate (EC No. 231 -626 -4), which is also an expected hydrolysis degradation product and therefore considered to be a relevant surrogate substance was negative in a guideline (OECD 476) mouse lymphoma assay with and without metabolic activation.

Justification for classification or non-classification

Based on the available data, TGEPO is not classified for mutagenicity according to Regulation (EC) No 1272/2008.