2-ethylhexyl mercaptoacetate

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl mercaptoacetate

EC Number:

231-626-4

EC Name:

2-ethylhexyl mercaptoacetate

Cas Number:

7659-86-1

Molecular formula:

C10H20O2S

IUPAC Name:

2-ethylhexyl 2-sulfanylacetate

Constituent 2

Reference substance name:

2-ethylhexyl thioglycolate

IUPAC Name:

2-ethylhexyl thioglycolate

Details on test material:

- Name of test material (as cited in study report): ethylhexyl thioglycolate
- Physical state: colourless liquid
- Expiration date of the lot/batch: September 22, 2000
- Storage condition of test material: 2-8 °C, protected from exposure to light

Test animals

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.

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.