(Z)-docos-13-enamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Jan - 10 Feb 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2002)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Ländlichen Raum und Verbraucherschutz

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

(Z)-docos-13-enamide

EC Number:

204-009-2

EC Name:

(Z)-docos-13-enamide

Cas Number:

112-84-5

Molecular formula:

C22H43NO

IUPAC Name:

(Z)-docos-13-enamide

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): Erucamide
- Physical state: solid
- Storage condition of test material: room temperature
- Expiration date of the lot/batch: 02 Jul 2012
- Analytical purity: 99.2%
- Lot/batch No.: 0821205214

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK, Ltd., Oxon, UK
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 18.3 – 22.9 g
- Housing: Animals were individually housed in Makrolon Type II cages, with wire mesh top suspended with granulated soft wood bedding
- Diet: Pelleted standard diet (Harlan Laboratories B.V. Horst, The Netherlands), ad libitum
- Water: (tap/filtered) water, ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 18 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

other: tetrahydrofuran

Concentration:

preliminary test: 10 and 25% (w/v)
main test: 5, 10 and 25% (w/v)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: highest concentration that was suitable for dosing: 25%
- Irritation: In the pre-test clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 7. At the tested concentrations, the animals did not show any signs of irritation on the ear skin. Signs of systemic toxicity were also not observed.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT:
- Name of test method: 3H-methyl thymidine incorporation
- Criteria used to consider a positive response: 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 3HTdR at least 3-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.

TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 5, 10, and 25% (w/v) in tetrahydrofuran. The application volume, 25 µL, was spread over the entire dorsal surface of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the vehicle alone (control animals). Five days after the first topical application, all mice were administered with 250 µL of 78.3 µCi/mL 3HTdR (corresponds to 19.6 µCi 3HTdR per mouse) by intravenous injection via a tail vein. Approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of Pentobarbital-Natrium. The draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were re-suspended in 5% trichloroacetic acid (approx. 3 mL) and incubated
at approximately + 4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then re-suspended in 5% trichloroacetic acid (1 mL) and transferred to plastic scintillation vials with 10 mL of ‘Ultima Gold’ scintillation and thoroughly mixed. The level of 3HTdR incorporation was then measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5% trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables.

Results and discussion

Positive control results:

A group of five animals for each dose group was treated with the positive control substance α-Hexylcinnamaldehyde as a solution in acetone:olive oil (4:1) at concentrations of 5,10 and 25% (w/v). A further control group of five animals was treated with the vehicle alone. The stimulation index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is 1.0, 1.21, 2.09 and 6.22 for the corresponding concentrations of 0, 5,10 and 25% (w/v) of the test substance. Therefore, α-Hexylcinnamaldehyde was considered to be a sensitizer at a concentration of 25% (w/v) under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

A concentration of 5% of the test substance shows a stimulation index (SI) of 0.81. 10% of the test substance shows a SI of 0.84 and the highest concentration of 25% test substance shows a SI of 0.70. Based on this result, the test substance can be considered as a non-sensitizer.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

The number of disintegrations per minute and lymph node (dpm/node) for the vehicle was 566.9. A concentration of 5% of the test substance shows a dpm/node of 456.8. 10% of the test substance shows a dpm/node of 474.5. The highest concentration of 25% test substance shows a dpm/node of 396.5.

Any other information on results incl. tables

Table 1: Calculation and Results of Individual Data

Test item concentration			DPM values measured	DPM-BG per animal (2 lymph nodes)^a)	S.I.^b)
% (w/v)	Group No.	Animal No.
---	BG I	---	65	---	---
---	BG II	---	21	---	---
0	1	1	1259	1216	---
0	1	2	2080	2037	---
0	1	3	822	779	---
0	1	4	1101	1058	---
0	1	5	622	579	---
5	2	6	741	698	0.6
5	2	7	1097	1054	0.9
5	2	8	1213	1170	1.0
5	2	9	696	653	0.6
5	2	10	1036	993	0.9
10	3	11	1239	1196	1.1
10	3	12	974	931	0.8
10	3	13	779	736	0.6
10	3	14	596	553	0.5
10	3	15	1372	1329	1.2
25	4	16	953	910	0.8
25	4	17	550	507	0.4
25	4	18	916	873	0.8
25	4	19	587	544	0.5
25	4	20	1174	1131	1.0

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

1 = Control Group

2 - 4 = Test Group

S.I. = Stimulation Index

^a) = values corrected for mean background value (BGI and BGII).

^b) = Stimulation Indices relative to the mean of the control group (Group 1)

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified

Executive summary:

In the study the test item Erucamide, dissolved in tetrahydrofuran, was assessed for its possible skin sensitization potential. For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25% (w/v). The animals did not show any signs of local skin irritation or systemic toxicity during the course of the study and cases of mortality were not observed. In this study Stimulation Indices (S.I.) of 0.81, 0.84 and 0.70 were determined with the test item at concentrations of 5, 10, and 25% (w/v) in tetrahydrofuran, respectively.

The test item Erucamide was not a skin sensitizer under the test conditions of this study.