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Diss Factsheets

Toxicological information

Skin sensitisation

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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
Chemical structure
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:
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:
preliminary test: 10 and 25% (w/v)
main test: 5, 10 and 25% (w/v)
No. of animals per dose:
5 females (nulliparous and non-pregnant)
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. As an applicable solution or suspension of the test item could not be obtained in the standard vehicles mentioned by this guideline, upon sponsor’s request, tetrahydrofuran was used as vehicle. The highest test item concentration where an applicable solution was obtained in tetrahydrofuran was 25% (w/v). Warming to 40°C and vortexing were necessary to formulate the test item.

In the pre-test, two mice were treated with test item concentrations of 10 and 25% (w/v) in THF 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.

- Irritation: No signs of irritation observed on the ear
- Systemic toxicity: No signs of systemic toxicity observed

The test item in the main study was assayed at 5, 10, and 25% (w/v). The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation.

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 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

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 positive control 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)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
Vehicle control (0%)
Key result
Parameter:
SI
Value:
0.81
Test group / Remarks:
Test substance (5%)
Key result
Parameter:
SI
Value:
0.84
Test group / Remarks:
Test substance (10%)
Key result
Parameter:
SI
Value:
0.7
Test group / Remarks:
Test substance (25%)

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:
other: CLP criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not classified
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.