Bis(2-ethylhexyl) 2,2'-thiobisacetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13th February 2008 - 19th February 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) 2,2'-thiobisacetate

EC Number:

246-131-9

EC Name:

Bis(2-ethylhexyl) 2,2'-thiobisacetate

Cas Number:

24293-43-4

Molecular formula:

C20H38O4S

IUPAC Name:

2-ethylhexyl 2-({2-[(2-ethylhexyl)oxy]-2-oxoethyl}sulfanyl)acetate

Details on test material:

- Name of test material (as cited in study report): Di (2-ethylhexyl) thiodiglycolate
- Physical state: clear yellow liquid
- Analytical purity: 97 %
- Lot/batch No.: 20705
- Expiration date of the lot/batch: 18 March 2008
- Storage condition of test material: room temperature in the dark over silica gel

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Limited, Bicester, Oxon, UK
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: individually in polypropylene cages with softwood bedding
- Diet (e.g. ad libitum): Certified Rat and Mouse diet
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 Feb 2008 To: 19 Feb 2008

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

5%, 10 %, 25 % and 50 % (v/v)

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
The mice were treated on three consecutive days by daily application of 25 µL of the undiluted test material or at a concentration of 50% (v/v) in acetone/olive oil 4:1.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
The number of radioactive disintegrations per minute per lymph node and the ratio of 3HTdR incorporation into lymph node cells relative to that recorded for the vehicle control was considered to express the proliferation response of the lymph node.

TREATMENT PREPARATION AND ADMINISTRATION:
On Day 6 all mice were injected with 3H-methyl thymidine (20 µCi per mouse). 5 hours later the auricular lymph nodes were excised and pooled for each experimental group. 3HTdR incorporation was measured by ß-scintillation counting using scintillation fluid (Optiphase 'Trisafe') and the Beckman LS6500 scintillation system.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

alpha-Hexylcinnamaldehyde at 5, 10 and 25 % v/v in ethanol/distilled water 7:3 gave stimulation indices of 1.38, 2.03 and 8.04, respectively.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: At 5, 10 and 20 % v/v test substance in acetone/olive oil 4:1 a stimulation index of less than 3 was recorded. At 50 % v/v test substance in acetone/olive oil 4:1 a stimulation index greater than 3 was recorded.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Results are given in Table 1.

Any other information on results incl. tables

Range finding test:

Hunched posture, tiptoe gait and occasional body tremors were noted in the animal treated with the undiluted test material.

This animal was humanely killed post dose on Day 2.

No signs of systemic toxicity were noted in the animal treated with the test material at 50 % v/v in acetone/olive oil.

Main test:

Body weight changes of the test animals were comparable to those of the control animals.

No signs of systemic toxicity and no mortalities were noted at 5, 15 and 25 % v/v.

At 50 % v/v of the test substance one animal showed hunched posture, tiptoe gait and fasciculations on Day 4; this animal was killed on Day 4 for humane reasons.

Table 1: Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% v/v) in acteone/olive oil 4:1	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	8492.58	1061.57	na	na
5	5090.96	636.37	0.60	Negative
10	6413.96	801.75	0.76	Negative
25	10568.68	1321.09	1.24	Negative
Vehicle	10377.16	1297.15	na	na
50	31911.55	3988.94	3.08	Positive

a= Disintegrations per minute/node obtained by dividing the disintegration per minute value by 8 (total number of lymph nodes)

b= Stimulation Index of 3.0 or greater indicates a positive result

na = not applicable

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the study results, the test substance Di-(2-ethylhexyl) thiodiglycolate may cause sensitization by skin contact according to EU classification criteria (Health Hazard, GHS H317).

Executive summary:

In a Local Lymph Node Assay (LLNA) with the test substance Di-2-EHTDG 16 female CBA/Ca mice were tested according to OECD TG 429. The test substance was applied at concentrations of 5%, 10%, 25% and 50% (v/v) in acetone/olive oil (4:1). Stimulation indices were <3 at concentrations up to 25% (v/v). At concentrations of 50% v/v and above, the test substance induced clinical signs of systemic toxicity in the animals. These animals were euthanized.

The stimulation index of 3.08 at 50% v/v test substance concentration is therefore considered to be sufficiently high to conclude that the test substance was sensitizing under the conditions of the test. Based on the study results, the test substance Di-2-EHTDG may cause sensitization by skin contact according to the GHS and EU classification criteria.