Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-04-14 to 2010-04-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): N,N’-Ethylenebis(stearamide)-commercial grade, formerly specified by CAS 110-30-5, but actually Amides, C16-C18 (even), N,N'-ethylenebis, EC 931-299-4
- Molecular formula: C38H76N2O2 (for CAS 110-30-5; molecular formula for actual substance cannot be specified due to UVCB-status)
- Molecular weight: 593.04 (for CAS 110-30-5; molecular weight for actual substance cannot be specified due to UVCB-status)
- Physical state: white crystalline solids
- Analytical purity: >85%
- Lot/batch No.: 033318
- Expiration date of the lot/batch: 19 January 2011 (allocated by NOTOX, 1 year after receipt of the test substance)
- Storage condition of test material: At room temperature in the dark, stable under storage conditions.
- Other: Source: Croda Europe Ltd

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: young adult, approx. 9 weeks old
- Weight at study initiation: variation was within +/- 20% of the sex mean (range 21 to 25 g)
- Housing: Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment. The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on Day 3.
- Diet (e.g. ad libitum): free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): free access to tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 (actual range: 18.9– 23.2)
- Humidity (%): 40-70 (actual range: 37 – 84)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 April 2010 To: 29 April 2010

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10, 25 or 50% w/w
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
Concentrations of 25% and 50% were tested in order to determine the highest test substance concentration to be used in the study, which should be well tolerated systemically and may give moderate irritation.
- Irritation: slight irritation (erythema and edema score of 1 of max. 4) was noted with 50% test substance

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM. The results were evaluated according to the Globally Harmonized System of Classification and
Labeling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labeling and packaging of substances and mixtures. Consideration was given to the EC3 value (the estimated test substance concentration that will give a SI =3).

TREATMENT PREPARATION AND ADMINISTRATION: In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The SI values calculated for the positive control substance concentrations 5, 10 and 25% were 1.7, 2.7 and 8.8 respectively. Based on the results, it was concluded that the Local Lymph Node Assay in the mouse as supplied by Janvier performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 10, 25 and 50% were 1.2, 1.3 and 0.9, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 210, 235 and 163 DPM, respectively. The mean DPM/animal value for the vehicle control group was 176 DPM.

Any other information on results incl. tables

Table 1: Individual animal scores

Group

concentration [% w/w]

animal

DPM

SI

1

0

1

256

1.45620023

2

300

1.70648464

3

137

0.77929465

4

65

0.36973834

5

121

0.68828214

mean

175.8

1

SEM

44

0.4

2

10

1

245

1.39362912

2

262

1.49032992

3

215

1.22298066

4

54

0.30716724

5

273

1.55290102

mean

209.8

1.19340159

SEM

40

0.4

3

25

1

211

1.20022753

2

132

0.75085324

3

137

0.77929465

4

413

2.34926052

5

280

1.592719

mean

234.6

1.33447099

SEM

52

0.4

4

50

1

147

0.83617747

2

219

1.24573379

3

118

0.67121729

4

178

1.01251422

5

153

0.87030717

mean

163

0.92718999

SEM

17

0.3

DPM: Disintegrations Per Minute

SI: Stimulation Index

SEM: Standard Error of Mean

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information