N,N'-ethane-1,2-diylbisoleamide

Skin sensitisation

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

Study period:

14 – 29 April 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance N,N’-Ethylenebis(stearamide). According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

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

Test material information

Constituent 1

Reference substance name:

Amides, C16-C18 (even) , N,N'-ethylenebis

EC Number:

931-299-4

Cas Number:

68390-94-3

Molecular formula:

not available UVCB

IUPAC Name:

Amides, C16-C18 (even) , N,N'-ethylenebis

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): N,N’-Ethylenebis(stearamide)-commercial grade
- Molecular formula: C38H76N2O2
- Molecular weight: 593.04
- Physical state: white crystalline solid
- Analytical purity: > 85%
- Lot/batch No.: 033318
- Expiration date of the lot/batch: 2011-01-19 (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: 21-25 g
- Housing: individual housing in labelled Macrolon cages (MI type; height 12.5 cm) containing sterilised 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: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 (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 sensitiser, 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)

Statistics:

Mean DPM values and standard errors per group were calculated.

Results and discussion

Positive control results:

The SI values calculated for the positive control substance at concentrations of 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)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

vehicle control

Results 2

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

10%

Results 3

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

25%

Results 4

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

50%

Results 5

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with the test substance at concentrations of 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 and mean DPM and SI values (n = 5 animals per group)

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:

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

Conclusions:

CLP: not classified