Bis(2-ethylhexyl) fumarate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 April to 1 July 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) fumarate

EC Number:

205-448-2

EC Name:

Bis(2-ethylhexyl) fumarate

Cas Number:

141-02-6

Molecular formula:

C20H36O4

IUPAC Name:

bis(2-ethylhexyl) but-2-enedioate

Details on test material:

- Name of test material (as cited in study report): dioctylfumarate
- Physical state: liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Species, strain: Mice, CBA/CaOlaHsd.
- Supplier: Harlan Winkelman GmbH, D-33176 Borchen.
- Sex, specification: Females, healthy young adult nulliparous animals.
- Age of the animals: About 8 weeks at the first administration.
- Weight range of the animals at the first application: 16.5–22.2 g.
- Number of animals: 5 animals/group (including spare animals):
15 animals for 3 test substance groups,
5 animals for the negative control group,
5 animals for the positive control group.
- Spare animals: The 5th animal of each group served as spare animal. It was treated in the same way as the other animals of its group.
Since all animals survived and all animals received the correct amount of 3HTdR all spare-animals were taken for the examination of the results.
The number of animals/group was then 5.

ENVIRONMENTAL CONDITIONS
- Hygiene: Optimal hygienic conditions.
- Room temperature: Average of 22.0 °C (continuous monitoring and recording, shortly interrupted due to calibration of the data recorder).
- Relative humidity: Average of 46.7 % (continuous monitoring and recording, shortly interrupted due to calibration of the data recorder).
- Light: Only artificial light from 6.00 a.m. to 6.00 p.m.
- Cages: Single caging. Makrolon cages type II, (22 × 16.5 cm ground area, 15 cm high).
- Feed: Altromin maintenance diet for rats and mice, item No. 1324 forte, ad libitum. Random samples of the feed are analysed for contaminants by Altromin, D-32791 Lage.
- Water: Tap water from Makrolon-bottles with stainless steel canules, ad libitum.
- Bedding material: Aspen wood chips, ABEDD Dominik Mayr KEG, A-8580 Köflach.
Germ reduction by autoclaving.
- Acclimatisation: 6 days

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

- Group A (low dose): 25 % (v/v) in acetone/olive oil
- Group B (mid dose): 50 % (v/v) in acetone/olive oil
- Group C (high dose): 100 % in acetone/olive oil

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
A range finding study was performed with two animals/concentration with following concentrations: 100 % and 50 %. In this study the animals were treated in the same manner as in a main study with 25 µL test substance on the dorsum of each ear on three consecutive days. Ear thickness and body weight were measured on Day 1 before the first administration and on Day 4 about 24 hours after the last administration.
In the range finding study none of the animals showed overt systemic toxicity or excessive local skin irritation. The observed little increase in ear thickness is known from our experience within the normal range and does indicate slight irritation at the most.

MAIN STUDY
Bias control
The individual animals were allocated to their groups by random numbers.
Test design
The test substance was administered in 3 concentrations to the dorsal surfaces of the ears of the animals of the test substance groups. In a manner identical to that of animals in the treatment groups animals of one negative control group and one positive control group were treated with AOO and HCA respectively. Each animal was treated for 3 consecutive days. 3 days after the last administration the proliferation of the lymphocytes of the draining lymph nodes was measured by the determination of the amounts of incorporated 3HTdR.
Incorporation of 3H-methyl thymidine in vivo
5 days after the first topical administration, each animal received 20 µCi 3HTdR by slow intravenous administration. The injection solution containing nominal 80 µCi/mL 3HTdR was prepared by the dilution of 1.152 mL 3HTdR (amersham pharmacia biotech, Kat. Nr.:TRA 310, batch 314, specific activity 2.0 Ci/mmol, radioactive concentration 1 mCi/mL, radiochemical purity 99.0%, thymine content 0.1%) with 13.248 mL PBS. Several minutes prior to 3HTdR administration the animals were kept restrained in Plexiglas-tubes and the tail veins were visualized by placing the tails in warm water. Then 250 µL of the injection solution were intravenously administered to each animal.
Preparation of single cell suspensions and determination of incorporated 3H-methyl thymidine
Approximately 5 hours after 3HTdR injection all animals were sacrificed by carbon dioxide asphyxiation and the draining auricular lymph nodes were rapidly excised. The lymph nodes of each group were pooled in PBS. A single cell suspension (SCS) of lymph node cells (LNC) was prepared by gentle mechanical disaggregation of the pooled lymph nodes through a 70 µm cell strainer. The SCSs were then transferred into centrifuge tubes and LNC were pelleted by centrifugation (4°C, max. 200 g, 10 min). Afterwards supernatants were removed by aspiration. Then the LNC were resuspended and washed twice with PBS. After the final washing the supernatants were removed leaving just a small volume (<0.5 mL) and macromolecules were precipitated by incubation with 5 % trichloroacetic acid (TCA) at 4 °C overnight. Each precipitate was pelleted by centrifugation (4 °C, max. 200 g, 10 min) and resuspended in 1 mL TCA. This suspension was transferred into scintillation vials containing 10 mL scintillation cocktail (Packard Bioscience: Ultima Gold, Bestellnr. 6013329) and 3HTdR incorporation was determined with a beta-scintillation counter (TriCarb 2200CA, Packard Instrument Co., protocol 4: single label 3H, dpm, AEC-quenchcurve in Ultima Gold from 21 June 2002).

Positive control substance(s):

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

Statistics:

Analysis of variance followed by the Scheffe-test: all data with means and standard deviations determined, comparisons between dosed groups and the negative control group (used for ear thickness)
t-test: all data with means and standard deviations determined, for comparison of two groups only (used for ear thickness).
P = 0.05 was chosen in each test. Two tailed tests were used.

Results and discussion

Positive control results:

No skin reactions were noted in all animals of the positive control group at each observation time.
Application of 25% HCA in AOO resulted in an SI of 5.8.
This result proves the sensitivity of the strain of animals used and the reliability of the experimental technique.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.7

Test group / Remarks:

low dose

Remarks on result:

other: see Remarks

Remarks:

The calculated stimulation indices (test substance/negative control ratio) were decisive for the grading of the potential of sensitisation: According to the guidelines the decision process with regard to a positive response includes a stimulation index of equal to or greater than 3, together with consideration of dose-response. group K (negative control): 1 group A (low dose): 0.7 group B (mid dose): 1.2 group C (high dose): 1.7 group P (positive control): 5.8

Results 2

Parameter:

SI

Value:

1.2

Test group / Remarks:

mid dose

Remarks on result:

other: see remarks

Remarks:

The calculated stimulation indices (test substance/negative control ratio) were decisive for the grading of the potential of sensitisation: According to the guidelines the decision process with regard to a positive response includes a stimulation index of equal to or greater than 3, together with consideration of dose-response. group K (negative control): 1 group A (low dose): 0.7 group B (mid dose): 1.2 group C (high dose): 1.7 group P (positive control): 5.8

Results 3

Parameter:

SI

Value:

1.7

Test group / Remarks:

high dose

Remarks on result:

other: see remarks

Remarks:

The calculated stimulation indices (test substance/negative control ratio) were decisive for the grading of the potential of sensitisation: According to the guidelines the decision process with regard to a positive response includes a stimulation index of equal to or greater than 3, together with consideration of dose-response. group K (negative control): 1 group A (low dose): 0.7 group B (mid dose): 1.2 group C (high dose): 1.7 group P (positive control): 5.8

Results 4

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: group K (negative control): 8462 group A (low dose): 6076 group B (mid dose): 10475 group C (high dose): 14174 group P (positive control): 49501

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Dioctylfumarate is regarded as not sensitising to skin in the LLNA according to the local lymph node assay according to OECD guideline 429, since the SIs of all examined test substance concentrations were clearly smaller than 3. According to the results of this study and to the Directive 2001/59/EC for classification, the test substance dioctylfumarate does not require classification as "R43 May cause sensitisation by skin contact".

Executive summary:

Aim

The local lymph node assay was performed to evaluate a possible skin sensitising potential of dioctylfumarate according to the OECD-Guideline 429, 24 April 2002.

Method

The test substance was used undiluted or solved in acetone:olive oil 4:1, v/v (AOO)and was administered to three groups of 5 female CBA/Ca mice. Administration was performed epicutaneously to the dorsal surface of both ears, once a day on three consecutive days. The volume administered was 25 µL per ear.

Concentrations used:

Group A (low dose): 25 % (v/v) solution of dioctylfumarate in AOO

Group B (mid dose): 50 % (v/v) solution of dioctylfumarate in AOO

Group C (high dose): 100 % solution of dioctylfumarate in AOO

Two groups with 5 animals each served as positive and negative controls. Both control substances were administered under identical conditions as the test substances.

The following solutions served as control substances:

Group P (positive control): 25 % (v/v) solution of hexyl cinnamic aldehyde in AOO

Group K (negative control): AOO

5 days after the first topical application, 3H-thymidine was intravenously administered to all mice via a tail vein. Approximately 5 hours later all animals were sacrificed, the draining auricular lymph nodes were excised, pooled for each group, and single cell suspensions were prepared. Then incorporation of 3H-methyl thymidine into the cells was determined (liquid scintillation counter) and compared with the negative controls. The stimulation index (SI) was calculated as the ratio of the disintegrations per minute (dpm) of the dosed groups or of the positive control group to the dpm of the negative control group.

Results

General

All animals survived till the end of the study. No adverse effects were noted in any animal.

Body masses and body mass gains were in the range to be expected from animals of the same strain, sex and age.

No skin irritating effects were observed in the test substance groups and both control groups throughout the whole study.

3H-thymidine incorporation, stimulation indices

The calculated stimulation indices (test substance/negative control ratio) were decisive for the grading of the potential of sensitisation: According to the guideline the decision process with regard to a positive response includes a stimulation index of equal to or greater than 3, together with consideration of dose-response.

The SIs of the tested test substance concentrations were 0.7 (low dose), 1.2 (mid dose) and 1.7 (high dose).

Positive control

The positive control substance led to a stimulation index of 5.8, thus demonstrating the validity of the experiment.

Conclusion

According to the OECD guideline 429, "Skin Sensitisation: Local Lymph Node Assay", dioctylfumarate is not regarded as a skin sensitiser in the LLNA.

According to the results of this study and to the Directive 2001/59/EC for classification, the test substance dioctylfumarate needs not to be labelled with “R43, may cause sensitisation by skin contact”.