2-Butenedioic acid (E)-, di-C8-18-alkyl esters

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

other: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Justification for type of information:

Justification for read-across is given in Section 13 of IUCLID.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

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:

Didodecyl fumarate

EC Number:

219-280-2

EC Name:

Didodecyl fumarate

Cas Number:

2402-58-6

Molecular formula:

C28H52O4

IUPAC Name:

didodecyl but-2-enedioate

Details on test material:

- Name of test material (as cited in study report): Didodecyl fumarate
- Physical state: solid
- Analytical purity: 93.8 area-%
- Lot/batch No.: 0008043725

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany.
- Age at study initiation: 8 weeks.
- Weight at study initiation: mean: 19.6 g.
- Housing: single housing in Makrolon cage, type II.
- Diet: STANRAB (P) SQC; SDS Special Diets Services. 67122 Altrip, Germany.
- Water: tap water ad libitum.
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C.
- Humidity: 30 - 70 %.
- Air changes: approx. 10 per hr.
- Photoperiod: 12 hrs dark/12 hrs light.

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

10 %, 25 %, 50%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS
- Compound solubility: the highest test item concentration, which could be technically used, was a 50 % (w/w) dilution in MEK.
- Irritation: no signs of local skin irritation were observed. One animal, treated with 25 %, showed white test item residues prior the 2nd application only.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: BrdU incoroporation measured in a photometer.
- Criteria used to consider a positive response: the proliferative response of the lymph node cells is expressed as the mean S.I. (Stimulation Index). The S.I. is derived by dividing the mean BrdU labeling index/mouse within each test substance group as well as for the positive control group by the mean BrdU labeling index for the vehicle group. A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled: first, that exposure to at least one concentration of the test item resulted in an incorporation of BrdU at least 1.6-fold or greater than that recorded in control mice, as indicated by the Stimulation Index and second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression. Furthermore, an index was calculated for the lymph node weight and - cell count as well as for the ear weight by dividing the mean values of the test item treated groups by the mean of the vehicle control group. For BALB/c mice, a cutoff-value for the lymph node cell count index of 1.55 was reported for a positive response (Ehling et al. (2005): An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay 2nd round. Toxicology, 212, 69–79.). Furthermore, according to OECD guideline 442B, an increase in ear weight exceeding the threshold value of 25 % was considered to be indicative for excessive local skin irritation.

TREATMENT PREPARATION AND ADMINISTRATION: each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 10, 25 and 50 % (w/w) in MEK. The application volume 25 µl/ear/day was spread over the entire dorsal surface (diameter ca. 8 mm) of each ear once daily for three consecutive days. The control test group of mice was treated with an equivalent volume of the relevant vehicle alone. The positive control test group of mice was treated with 25 % α-hexyl cinnamaldehyde dissolved in MEK. For injection BrdU was dissolved in DPBS (10 mg/ml) before administration and stored in a refrigerator until use. Four days after the first topical application (day 5) 0.5 ml of BrdU solution (5mg/per mouse/injection) was intraperitoneally injected. Approximately 24 hours after treatment with BrdU the mice were sacrificed and the draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through a 70 µm nylon mesh. The lymph node cells were re-suspended in 6 ml DPBS. After cell count with a cell counter (Casy Cell Counter, Innovatis), cell suspensions of 100 000 cells/ml were adjusted.

Positive control substance(s):

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

Statistics:

A Mann-Whitney-U test for non-parametric comparison was applied as statistical test. Statistical significance was set at the five per cent level (p < 0.05). However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:

25 % α-hexyl cinnamaldehyde: S.I. = 5.0

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.9

Test group / Remarks:

50 % test group

Remarks on result:

other: mean value

Results 2

Parameter:

SI

Value:

0.9

Test group / Remarks:

25 % test group

Remarks on result:

other: mean value

Results 3

Parameter:

SI

Value:

1.2

Test group / Remarks:

10 % test group

Remarks on result:

other: mean value

Results 4

Parameter:

SI

Value:

1

Test group / Remarks:

vehicle control

Remarks on result:

other: mean value

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
- The measured lymph node weights and cell counts of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in lymph node weights or cell counts was not observed in any of the test item treated groups in comparison to the vehicle control group. In the positive control a statistically significant and biological relevant increase was observed.
- The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant or biological relevant increase in ear weights was not observed in any test item treated group in comparison to the vehicle control group.
According to OECD guideline 442B, an increase in ear weight exceeding the threshold value of 25 % was considered to be indicative for excessive local skin irritation. This threshold was not exceeded in any test item treated group. In the positive control a statistically significant and biological relevant increase was observed.

OTHER OBSERVATIONS
- No deaths occurred during the study period.
- No symptoms of systemic toxicity were observed during the study period.
- No local findings (ears) were observed in the test item dose groups. Scaling and incrustations were noted in the positive control.

BODY WEIGHTS: the body weight of the animals recorded prior to the first application and prior to sacrifice were within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

other: not classified for skin sensitisation according to CLP Regulation (EC) No.1272/2008

Conclusions:

Non sensitising

Executive summary:

The skin sensitision potential of the category member substance was assessed in the Local Lymph Node Assay - BrdU-ELISA according to the OECD Guideline 442B. Five female CBA mice per group were treated with the test item at concentrations of 10, 25 and 50 % (w/w) for three consecutive days. Four days after the first topical application 0.5 ml of BrdU solution (5mg/per mouse/injection) was intraperitoneally injected. Approximately 24 hours after treatment with BrdU the mice were sacrificed and the draining lymph nodes were rapidly excised and pooled for each animal. Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation and the lymph node cells were re-suspended in DPBS. After cell count with a cell counter, cell suspensions of 100 000 cells/ml were adjusted.

The animals did not show any signs of systemic toxicity or local findings during the course of the study and no cases of mortality were observed. A statistically significant or biologically relevant increase in BrdU incorporation and also in lymph node weight, cell count and ear weight measurement was not observed in any of the test item dose groups in comparison to the vehicle control group. Furthermore, the cut-off-value for a positive response regarding the ear weight (25 % increase) was not exceeded in any dose groupIn the positive control scaling and incrustations were observed. A statistical and biological relevant increase in BrdU labelling, lymph node weight, lymph node cell count and ear weight measurement was determined in the positive control.

None of the tested concentrations resulted in a 1.6 -fold or greated increase in incorporation of BrdU compared with concurrent controls as indicated by the Stimulation Index (S.I.); S.I. of 1.2, 0.9 and 0.9 were determined with the test item at concentrations of 10, 25 and 50 % (w/w) in MEK.

An EC1.6 value could not be determined since all S.I. obtained were below the threshold of 1.6.

The test item is thus not a skin sensitiser under the test conditions of this study.