Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study with certificate and no deviations. Complete test article characterization available. Reliability was changed from "1" to "2" according to the ECHA guidance document "Practical guide 6: How to report read-across and categories".

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (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

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
IUPAC Name:
docos-13-enamide
Test material form:
other: solid, not further specified
Details on test material:
- Name of test material (as cited in study report): Erucamide
- Substance type: Fatty acid amide
- Physical state: solid
- Analytical purity: 99.2%
- Lot/batch No.: 0821205214
- Expiration date of the lot/batch: 02 July 2012
- Stability under test conditions: Not indicated by the sponsor.
- Storage condition of test material: At room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst/The Netherlands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 18.3-22.9 g
- Housing: Single in Makrolon Type II cages with wire mesh top (EHRET GmbH, 793002 Emmendingen, Germany) on granulated soft wood bedding (Rettenmaier & Soehne GmbH + Co. KG, 73494 Rosenberg, Germany)
- Diet: Pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst/The Netherlands), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 18-65
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
other: tetrahydrofuran
Concentration:
0, 5, 10 and 25% w/v (highest applicable test item concentration)
No. of animals per dose:
5 females (nulliparous and non-pregnant)
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: As an applicable solution or suspension of the test item could not be obtained in the standard vehicles mentioned in the guideline, tetrahydrofuran was used as vehicle upon sponsor's request. The highest applicable test item concentration obtained in tetrahydrofuran was 25% (w/v). Warming and vortexing were necessary to formulate the test item.
- Irritation: To determine the highest non-irritant test item concentration, a pre-test was performed in two animals. Two mice were treated with test item concentrations of 10 and 25% (w/v) each on three consecutive days. 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 to the ear skin. Signs of systemic toxicity were not observed, either. Thus, the test item in the main study was assayed at 5, 10 and 25% (w/v). The top dose is the highest technically achievable concentation whilst avoiding systemic toxicity and excessive local irritation.
- Lymph node proliferation response: The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (Stimulation Index; S.I.). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: 1) Exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR of at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index. 2) 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:
The test item was placed into a volumetric flask on a tared balance and tetrahydrofuran was quantitatively added. The different test item concentrations were prepared serially. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied.

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 (8 mm²) 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 euthanised by intraperitoneal injection of Pentobarbital-Natrium (Release®, WDT, 30827 Garbsen, Germany).

Mortality/viability was recorded once daily from experimental start to necropsy. Body weights were recorded prior to the first application and prior to treatment with 3HTdR. Clinical signs (local/systemic) were recorded within 1 hour after each application, and 24±4 hours after the first and second application as well as on the day of preparation.

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 resuspended 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 resuspended in 5 % trichloroacetic acid (1 ml) and transferred to plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany) and thoroughly mixed.

The level of 3HTdR incorporation was then measured on a ß-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany). Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The ß-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 of the body weights were calculated.

Results and discussion

Positive control results:
Results of the GLP Positive Control (Experiment performed in November 2009):
Positive control substance: alpha-Hexylcinnamaldehyde
Vehicle: acetone:olive oil (4+1)

Vehicle: S.I. 1.0; 498.1 DPM per lymph node
5%: S.I. 1.21; 600.9 DPM per lymph node
10%: S.I. 2.09; 1042.8 DPM per lymph node
25%: S.I. 6.22; 3096.1 DPM per lymph node

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Vehicle: 1.00 5%: 0.81 10%: 0.84 25%: 0.70
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Vehicle: 566.9 DPM per lymph node 5% : 456.8 DPM per lymph node 10%: 474.5 DPM per lymph node 25%: 396.5 DPM per lymph node

Any other information on results incl. tables

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)

Observations:

The EC3 value could not be calculated since all obtained SI's were below 3.

No deaths occurred during the study period.

The animals did not show any signs of local skin irritation or systemic toxicity during the course of the study.

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

CONCLUSION:

The test item was does not have to be considered as skin sensitiser under the conditions of this study.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test item Erucamide was not a skin sensitizer under the test conditions of this study.
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.