2-ethylhexyl mercaptoacetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

EHTG was negative for mutagenicity in a standard bacterial mutagenicity test with and without metabolic activation (Molinier, 1993). This study followed OECD test guideline 471. Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.EHTG did not induce mutagenicity in Salmonella bacterial strains with or without metabolic activation. The positive controls responded as expected.

 

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 n° 476 Guideline and in compliance with the Principles of Good Laboratory Practice (Sire, 2010). 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 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.

 

Handbook review presented the results of a chromosomal aberration assay consisting in investigations of the effects of the substance on Chinese Hamster V79 cells in vitro (BG Chemie, 1993). Three concentrations were tested: 75, 150 and 300 µg/ml with metabolic activation; 37.5, 75 and 150 µg/ml without metabolic activation. Increases in chromosome aberrations or in polyploid cells were seen only at the highest concentration tested both in the absence and presence of metabolic activation. Nevertheless, effects observed at this concentration were considered to be due to a general cytotoxic effect rather than a clastogenic effect.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 28 April to 24 June 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

This test enables the detection of base-pair substitution and frameshift mutagens. Mutagenic substances can induce reversion in histidine-deficient strains, which are then able to grow and form colonies in a histidine-limited medium, while non-revertants can not.

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA102, TA1535 & TA1537

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Without S9: 2.5, 5, 10, 20 & 50 µg/plate
With S9: 250, 500, 1000, 2000 & 4000 µg/plate (1st assay); 31.25, 62.5, 125, 250 & 500 µg/plate (2nd assay)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not reported but commonly used for this type of study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9 mix: · 1 µg/plate of sodium azide (NaN3): TA 1535 and TA 100 strains, · 50 µg/plate of 9-Aminoacridine (9AA): TA 1537 strain, · 0.5 µg/plate of 2-Nitrofluorene (2NF): TA 98 strain, · 0.5 µg/plate of Mitomycin C (MMC): TA 102 strain. With S9 mi

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation
- Type and identity of media:
Direct plate incorporation method:
Molten agar containing traces of histidine and biotin and maintained at 45°C. Then Petri plate containing minimum medium.

Preincubation method:
Minimum agar plate.

DURATION
- Preincubation period: 14 hours
- Exposure duration: 48 to 72 hours

SELECTION AGENT (mutation assays): Histidine

NUMBER OF REPLICATIONS: 2 independent tests, using 3 plates/concentration

DETERMINATION OF CYTOTOXICITY
- other: observation whether the bacterial lawn is sparse and whether the number of colonies is decreased.

Evaluation criteria:

Positive response if:
- a reproducible and significant (if p <= 0.05) dose relationship, and/or
- a reproducible and significant (doubling in the number of revertants) increase

Test valid because:
- Results of the negative and vehicle controls in the range of corresponding historical data, and
- Results of the positive control higher than results of negative and vehicle controls and in the range of corresponding historical data

Statistics:

Linear regression analysis

Species / strain:

S. typhimurium, other: TA98, TA100, TA102, TA1535 & TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9: >= 50 µg/plate|with S9: >= 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and solvent control results were equivalent to those usually obtained in the laboratory. The number of revertants induced by the
positive controls was higher than the controls, indicating the sensitivity of the test.
RANGE-FINDING/SCREENING STUDIES:
Precipitation was observed from 500 µg/plate in the first cytotoxicity test conducted without S9. It was not observed in the corresponding test
conducted with S9.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and solvent control results were equivalent to those usually obtained in the laboratory. The number of revertants induced by the
positive controls was higher than the controls, indicating the sensitivity of the test.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Mean Revertants per Plate – Direct plate incorporation method

Test Material	TA-1535		TA-1537		TA-102		TA-98		TA-100
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Nonactivation
Negative Control	11	2	12	2	160	17	18	3	92	4
Solvent Control	11	2	6	2	219	8	12	4	71	16
2.5 µg/plate	13	1	9	1	196	16	18	1	80	5
5 µg/plate	13	3	11	2	199	22	13	3	81	4
10 µg/plate	9	3	7	2	245	20	14	4	80	13
20 µg/plate	11	2	7	1	222	33	13	1	84	4
50 µg/plate	5	1	8	2	226	8	17	2	86	12
Positive Control	188	36	149	13	1005	236	109	10	323	23
Activation
Solvent Control	13	1	10	1	297	20	25	3	108	7
250 µg/plate	13	4	8	1	261	14	24	4	101	12
500 µg/plate	10	5	7	2	278	35	22	7	119	7
1000 µg/plate	11	3	6	1	304	23	22	6	115	6
2000 µg/plate	13	2	8	2	320	27	20	5	105	7
4000 µg/plate	11	1	10	1	324	31	23	3	100	28
Positive Control	252	35	147	9	895	66	1767	131	1573	43

Table 2: Mean Revertants per Plate – Preincubation method

Test Material	TA-1535		TA-1537		TA-102		TA-98		TA-100
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Nonactivation
Negative Control	10	4	12	3	261	8	15	1	79	5
Solvent Control	7	3	13	2	210	8	15	5	69	8
2.5 µg/plate	9	3	13	3	254	23	14	4	84	4
5 µg/plate	7	1	10	3	258	10	18	5	84	8
10 µg/plate	6	3	13	2	264	11	17	2	74	7
20 µg/plate	8	0	13	4	238	30	14	4	82	10
50 µg/plate	7	2	9	2	353	15	20	2	76	7
Positive Control	232	5	185	21	1676	185	133	12	296	23
Activation
Solvent Control	9	4	12	1	414	23	25	1	110	19
31.25 µg/plate	13	4	9	2	383	11	24	1	98	13
62.5 µg/plate	10	1	13	3	354	24	30	8	106	6
125 µg/plate	12	4	9	3	355	41	24	5	91	4
250 µg/plate	11	0	9	1	348	7	30	3	95	4
500 µg/plate	4	3	6	3	344	26	14	2	22	39
Positive Control	158	5	222	9	871	76	1710	75	1397	27

Conclusions:

Interpretation of results (migrated information):
negative

The test substance was found to be negative in the in vitro genotoxicity Ames assay both in the presence and absence of metabolic activation.

Executive summary:

The genotoxicity of the substance was determined in a reverse mutation assay conducted in vitro with salmonella typhimurium TA 1535, TA 1537, TA 102, TA 98 and TA 100 according to the OECD Guideline 471. Concentrations tested ranged from 2.5 to 50 µg/plate and from 31.5 to 4000 µg/plate in the absence of S9 and in the presence of S9, respectively. The test substance did not induce any significant increase in the number of revertants, with or without S9 mix, in any of the 5 strains.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 May 2008 - 10 Feb.2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP 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:

mammalian cell gene mutation assay

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 n° 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 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 cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data from Handbook

Principles of method if other than guideline:

Methof used and Guidelines followed were not reported in the review document.

GLP compliance:

not specified

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:

S9-mix from Aoclor 1254-induced rat

Test concentrations with justification for top dose:

75, 150 and 300 µg/ml with metabolic activation
37.5, 75 and 150 µg/ml without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Details on test system and experimental conditions:

METHOD OF APPLICATION: no data

DURATION
- Exposure duration: 5 hours
- Expression time (cells in growth medium):
At the highest concentration: 7, 18 and 28 hours
At the 2 lowest concentrations: 18 hours


NUMBER OF REPLICATIONS: 4 cultures per concentration


NUMBER OF CELLS EVALUATED:
100 metaphases per culture

DETERMINATION OF CYTOTOXICITY
No data:


OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

no data

Statistics:

no data

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

Effects at the highest dose tested (with and without S9) are deemed to be due to cytotoxic effects

Cytotoxicity / choice of top concentrations:

other: ambiguous (indicated at the highest dose tested (with and without S9) but no specific data presented)

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

not specified

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: cytototoxicity evaluation
In an initial study, thioglycolic acid 2-ethylhexyl ester did not cause any increase in chromosome aberrations or in polyploid cells. These findings were fully confirmed for the 18- and 28-hour time points in a repeat of this study. However, there was a slight but statistically significant increase in the proportion of aberrant chromosomes in the preparations processed after 7 hours both with and without metabolic activation, which when combined with the first study was only significant without metabolic activation. These findings were confirmed in two additional studies, with 150 µg/ml (without activation) and 300 µg/ml (with activation). It could be assumed that a general cytotoxic effect rather than a clastogenic effect was responsible for these results.

Conclusions:

Handbook review presented the results of a chromosomal aberration assay. Some increases in chromosome aberrations or in polyploids cells were reported at the highest dose tested (both in the absence and presence of metabolic activation). Handbook review reported that according to the authors of the primary study, it could be assumed that a general cytotoxic effect rather than a clastogenic effect was responsible for these results.

Executive summary:

Handbook review presented the results of a chromosomal aberration assay consisting in investigations of the effects of the substance on Chinese Hamster V79 cells in vitro. Three concentrations were tested: 75, 150 and 300 µg/ml with metabolic activation; 37.5, 75 and 150 µg/ml without metabolic activation. Increases in chromosome aberrations or in polyploid cells were seen only at the highest concentration tested both in the absence and presence of metabolic activation. Nevertheless, effects observed at this concentration were considered to be due to a general cytotoxic effect rather than a clastogenic effect.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

EHTG was given to both male and female ICR mice as a single dose by intraperitoneal injection in a standard micronucleus study (OECD Guideline 474) (BioReliance, 1998). Based on the results of a preliminary toxicity assay, the dose levels for the definitive study were: 175, 350 and 700 mg/kg (males) and 225, 450 and 900 mg/kg (females).Five mice per sex from each group were sacrificed at 24 and 48 hours after dose administration. A total of 2000 polychromatic erythrocytes from bone-marrow suspension were scored per mice for the presence of micronuclei. The proportion of PCEs to total erythrocytes was recorded per 1000 erythrocytes. There were no signs of toxicity in the low dose group animals. In the mid-dose group animals signs of toxicity included lethargy and piloerection in 5/5 males and females and piloerection in 3/5 females. In the high dose group animals lethargy was observed in 15/15 males and females, hunched position in 7/15 males and 13/15 females. There was no effect on PCE/NCE ratio and the frequency of micronucleated PCE compared to controls at any dose level. The positive controls responded as expected.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, MD, USA
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: Males: 26.1-32.2 g; Females: 23.3-29.2 g
- Assigned to test groups randomly: yes, according to a computer-generated program, based on distribution according to body weight
- Fasting period before study: not reported
- Housing: Mice of the same sex were housed up to five per cage
- Diet: certified laboratory rodent chow ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.5 +/- 3
- Humidity (%): 50 +/- 20
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: The study initiation date was indicated to be on 5 October 1998. The study completion date was not indicated.

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: knowledge of the test substance (TS) by the company requestinq the study and compability of the vehicle with the test animals
- Concentration of test material in vehicle: TS was soluble in corn oil at 100 mg/mL, the maximum concentration tested. Dosing concentrations were delivered to the test system as solutions.

Details on exposure:

Solutions were administered by intraperitoneal injection at a constant volume of 20 mL/kg bw.

Duration of treatment / exposure:

single administration

Frequency of treatment:

single administration

Post exposure period:

24 h for vehicle and all treated groups, additionally 48 h for vehicle and highest tested concentration group

Remarks:

Doses / Concentrations:
Male: 175, 350 & 700 mg/kg bw. Female: 225, 450 & 900 mg/kg bw
Basis:

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): not reported but substance commonly used as positive control
- Route of administration: intraperitoneal injection
- Doses / concentrations: 50 mg/kg bw

Tissues and cell types examined:

Bone marrow cells of the femurs

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Doses selected according to the results of preliminary toxicity assay (pilot and toxicity study). In the toxicity assay, male and female mice were dosed at 150, 300, 500 or 800 mg/kg. Mortality was observed in 2/5 male mice at 800 mg/kg. Clinical signs following dose administration included
lethargy and piloerection in all male and female mice at 500 and 800 mg/kg. Due to mortality observed only in male high dose group, the high dose
for the micronucleus test was set at 700 mg/kg for male mice and at 900 mg/kg for female mice.

DETAILS OF SLIDE PREPARATION:
Bone marrow cells were transferred to a capped centrifuge tube containing 1 mL fetal bovine serum. After centrifugation, serum with the remaining
cell pellet was recovered. Cells were resuspended and suspension was spread onto glass slide. Two to four slides were prepared from each mouse.
The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS:
Slides were randomly coded. Using oil immersion, 2000 polychromatic erythrocytes were scored for the presence of the micronuclei.

Evaluation criteria:

TS was considered to induce a positive response if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically higher than the respective control at any sampling time.
The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated.
The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.

Statistics:

Statistical significance using the Kastenbaum-Bowman tables based on the binomial distribution.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Mortality was observed in 1/15 female mice receiving 900 mg/kg bw. Clinical signs following dose administration included: lethargy in male mice at 350 and 700 mg/kg bw, piloerection and hunched position in male mice at 700 mg/kg bw, lethargy and piloere

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Dose selection assays (see Table 1 in the field Remarks on results):
- Pilot study:
Mortality was observed in 1/2 male mice at 1000 mg/kg bw and in all animals of both sexes at 2000 mg/kg bw.
Clinical signs following dose administration included lethargy and piloerection in male mice at 1000 mg/kg bw.
No significant effects of treatment on bodyweight were observed.
- Toxicity assay:
Mortality was observed in 2/5 male mice at 800 mg/kg bw. Clinical signs following dose administration included lethargy and piloerection in all male and female mice at 500 and 800 mg/kg bw.

Micronucleus assay (see Table 2 in the field Remarks on results):
Moderate reductions (up to 23%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in male and female treated groups at 48 hours after treatment relative to the respective vehicle control groups. These reductions suggest bioavailability and toxicity of the test article to the bone marrow.
A statistically significant increase in micronucleated polychromatic erythrocytes was observed in males of the highest treated group at 24 hours harvest time relative to the vehicle control group. The number of micronuclei observed in this group was within historical solvent control range (0-8 micronuclei/1000 PCE). No significant increases in micronucleated polychromatic erythrocytes were observed at 24 and 48 hours after dose administration in any other treated groups.

Table 1 Preliminary toxicity results

Test design	Treatment	Mortality ratio
		Males	Females
Phase I: Pilot study	TS at 1 mg/kg bw	0/2	-
	TS at 10 mg/kg bw	0/2	-
	TS at 100 mg/kg bw	0/2	-
	TS at 1000 mg/kg bw	1/2	-
	TS at 2000 mg/kg bw	5/5	5/5
Phase II: Toxicity assay	TS at 150 mg/kg bw	0/5	0/5
	TS at 300 mg/kg bw	0/5	0/5
	TS at 500 mg/kg bw	0/5	0/5
	TS at 800 mg/kg bw	2/5	0/5

-          test not conducted

Table 2 Micronucleus assay results

Treatment	Harvest time (h)	Sex	PCE/total erythrocytes (mean +-/ sd)	Number of micronucleated PCE per 1000 PCE (mean+-/ sd)
Vehicle control Corn oil at 20 mL/kg	24	M	0.47 +/- 0.05	0.4 +/- 0.42
	24	F	0.49 +/- 0.05	0.7 +/- 0.57
	48	M	0.50 +/- 0.05	0.4 +/- 0.42
	48	F	0.52 +/- 0.02	0.1 +/- 0.22
TS at 175 mg/kg	24	M	0.52 +/- 0.05	0.4 +/- 0.22
TS at 225 mg/kg	24	F	0.50 +/- 0.07	0.3 +/- 0.45
TS at 350 mg/kg	24	M	0.53 +/- 0.02	0.3 +/- 0.45
TS at 450 mg/kg	24	F	0.54 +/- 0.05	0.6 +/- 0.65
TS at 700 mg/kg	24	M	0.56 +/- 0.04	1.8 +/- 1.20
	48	M	0.39 +/- 0.04	0.1 +/- 0.22
TS at 900 mg/kg	24	F	0.50 +/- 0.08	0.7 +/- 0.57
	48	F	0.40 +/- 0.04	0.7 +/- 0.45
Positive control CP at 50 mg/kg	24	M	0.44 +/- 0.03	31.3 +/- 7.47
	24	F	0.44 +/- 0.02	26.4 +/- 7.54

PCE: polychromatic erythrocytes

Conclusions:

Interpretation of results (migrated information): negative
A statistically significant increase in micronucleated polychromatic erythrocytes was observed in males of the highest treated group at 24 hours
harvest time relative to the vehicle control group. The number of micronuclei observed in this group was within historical solvent control range (0-8 micronuclei/1000 PCE). No significant increases in micronucleated polychromatic erythrocytes were observed at 24 and 48 hours after dose administration in any other treated groups. Therefore, the test substance was concluded to be negative in the micronucleus test with mice.

Executive summary:

EHTG was given to both male and female ICR mice as a single dose by intraperitoneal injection in a standard micronucleus study (OECD Guideline 474). IBased on the results of a preliminary toxicity assay, the dose levels for the definitive study were: 175, 350 and 700 mg/kg (males) and 225, 450 and 900 mg/kg (females). Five mice per sex from each group were sacrificed at 24 and 48 hours after dose administration. A total of 2000 polychromatic erythrocytes from bone-marrow suspension were scored per mice for the presence of micronuclei. The proportion of PCEs to total erythrocytes was recorded per 1000 erythrocytes. There were no signs of toxicity in the low dose group animals. In the mid-dose group animals signs of toxicity included lethargy and piloerection in 5/5 males and females and piloerection in 3/5 females. In the high dose group animals lethargy was observed in 15/15 males and females, hunched position in 7/15 males and 13/15 females. There was no effect on PCE/NCE ratio and the frequency of micronucleated PCE compared to controls at any dose level. The positive controls responded as expected. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

2-EHTG was found to be 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 concluded to be negative in the micronucleus test with mice.

Justification for classification or non-classification

None of the available test results in vitro showed that substance treatment induced mutagenic effects. The micronucleus test result was negative. The susbtance should not be classified for genotoxicity.