2-Butenedioic acid (2E)-, di-C12-14 (even numbered)-alkyl esters

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline Study. In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

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:

2-Butenedioic acid (2E)-, di-dodecyl ester

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

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.. 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.
- 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

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Vehicle control: mean: S.I. = 1.0 10% test item: mean: S.I. = 1.2 25% test item: mean: S.I. = 0.9 50% test item: mean: S.I. = 0.9 A test item is regarded as a sensitizer in the LLNA if exposure to one or more test item concentration results in a 1.6-fold or greater increase in incorporation of BrdU compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 1.6 is referred to as the EC1.6 value. An EC1.6 value could not be determined as all S.I.s obtained were below the threshold of 1.6

Any other information on results incl. tables

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. 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.

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.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not sensitising
DSD: not sensitising