Fatty acids, coco derivs., reaction products with glycine, potassium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation, other

Remarks:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

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:

dipotassium 2-dodecanamidoacetate 2-tetradecanamidoacetate

EC Number:

620-582-5

Cas Number:

301341-58-2

Molecular formula:

R-CO-NH-CH2-COOK (Note: "R-CO-" is a fatty acid residue)

IUPAC Name:

dipotassium 2-dodecanamidoacetate 2-tetradecanamidoacetate

Test material form:

solid - liquid: suspension

Specific details on test material used for the study:

Lot no: 110825
Date of Expiration: 25.08.2014
Appearance: white powder

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

strain: Balb/c:AnCrl

Study design: in vivo (LLNA)

Vehicle:

other: acetone/olive oil (5:1 v/v)

Concentration:

1, 10 or 25%

No. of animals per dose:

6

Details on study design:

The original LLNA is based on the use of radioactive labelling for the measurement of cell proliferation, however other endpoints for the assessment of proliferation are also authorized by the OECD guideline 429. The non-radioactive method is an alternative and was validated in European inter-laboratory validation studies under the participation of Fraunhofer ITEM. One important aspect of the validation process of the alternative non-radioactive cell counting was comparing the sensitivity of the radioactive and non-radioactive method. All results found to be positive by the cell count method were also found to be positive by the radioactive assay, i.e. the modified LLNA is reliable and discriminates between sensitizing and irritating properties of a test item. Therefore, it avoids false positive results of the radioactive LLNA by including the measurement of acute skin reaction as an additional parameter for irritating properties of a test item and this modified method is considered to be suitable for regulatory purposes.
In this study groups of young adult female mice (Balb/c:AnCr l) were exposed topically on the dorsum of both ears to 25 uL of test item, reference item, or vehicle. Treatment was performed daily for 3 consecutive days.
The highest concentration of 50 % could not be applied as scheduled as it was not possible to suspense the appropriate amount of test item.
The mice were killed on day 4 and small pieces were stamped from each ear and weighed. The draining lymph nodes were excised and pooled for each animal. The lymph nodes were weighed and a single cell suspension was prepared by mechanical disaggregation. Cell counts were measured.

Positive control substance(s):

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

Statistics:

yes

Results and discussion

Positive control results:

HCA was confirmed as a skin sensitiser.

In vivo (LLNA)

Results

Key result

Parameter:

other: differentiation index (see below)

Remarks on result:

other: see below

Any other information on results incl. tables

In this study the differentiation index (DI) was determined, which describes the relation between skin-draining lymph node cell activation and skin inflammation:

DI > 1 indicates a chemically induced skin sensitization

0 < DI < 1 demonstrates an irritant potency of the test item.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed no increase at concentrations of 1% and 25%, but a significant increase of cells counts and lymph node weights at a concentration of 10%. The DI was < 1 at 1% and 25% and 1.9 at 10%, which was mainly due to the response of one animal. Thus, the DI was not as clearly increased as observed for the reference item and not confirmed at 1% and/or 25% tested dose. In conclusion, as there was no dose-response the test item should is not assessed to be a skin sensitizer.

Executive summary:

In a study performed to OECD Guideline 429 with GLP compliance, female mice were exposed topically on the dorsum of both ears to the test item at concentrations of 1%, 10% and 25% in acetone/olive oil (AOO) for three consecutive days.Control mice were treated with AOO (vehicle control) or with the reference item hexyl cinnamic aldehyde (HCA) in AOO (positive control) at concentrations of 10% and 30%. Mice were sacrificed after three days, small pieces were stamped from each ear, and weighed. The draining lymph nodes were excised, weighed, and single cell suspensions were prepared. Cell counts of the lymph node cell suspensions were measured, and based on significant responses in ear swelling and increases in lymph node cell counts a differentiation index (DI) was calculated for test item and reference item. The DI describes the relation between skin-draining lymph node cell activation (lymph node cell count index) and skin inflammation (ear weight index), with a DI > 1 indicating a chemically induced allergic reaction (skin sensitization), whereas 0 < DI < 1 demonstrates an irritant potency of the tested substance.

HCA as reference item and dosed at 10% and 30% showed significant increases in lymph node weights and lymph node cell counts in comparison to vehicle controls and thus was tested as skin sensitizing (DI > 1).

The test item showed no increase at concentrations of 1% and 25%, but significant increases of cell counts and lymph node weights at a concentration of 10%. At 1% and 25% the DI was < 1, and 1.9 at 10%, which was mainly due to the response of one animal. Nevertheless, the DI was not as obviously increased as observed for the reference item and not confirmed at 1% and 25%. According to the OECD Guideline 429, other aspects such as the strength of the dose-response and the positive control responses may also be used when determining whether a borderline result is declared positive. In particular the proliferation of cells in the lymph node is considered to be proportional to the dose and to the potency of the applied allergen.

In conclusion, as there was no dose-response the test item should is not assessed to be a skin sensitizer.