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

Endpoint summary

Description of key information

Skin sensitisation (OECD 429): not sensitising, based on read-across from Amides, C16-C18 (even), N,N’-ethylenebis

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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.

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)

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

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.

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.

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

vehicle control

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

50%

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.

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

Interpretation of results:

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

Conclusions:

CLP: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

There is no data available on the skin sensitisation potential of Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis. In order to fulfil the standard information requirements set out in Annex VII, 8.3, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from a structurally related substance is conducted.

A detailed justification for the grouping of chemicals and read-across is provided in the Sections 7.1 and 13.

There exists one reliable study on the skin sensitisation potential of the structurally related substance Amides, C16-C18 (even), N,N'-ethylenebis. Therefore, read-across based on the analogue approach is performed to cover this endpoint.

A Local Lymph Node Assay was performed in mice with the test substance at concentrations of 10, 25 and 50% in acetone/olive oil (4:1 v/v) according to OECD guideline 429 and in compliance with GLP (Stitzinger, 2010). The animals (5 per group) were epidermally treated (25 µL/ear) with the respective concentrations of the test substance or vehicle alone for 3 consecutive days, and skin irritation was assessed on Day 3. The slight erythema observed on the ears of animals treated with 50% of test substance was considered not to have a toxicologically significant effect on the activity of the lymph nodes. On Day 6, auricular lymph nodes were excised, and their relative size and the incorporated radioactivity of the injected ³H-methyl thymidine were assessed.The mean disintegration per minute (DPM) values for the single cell suspensions of each animal were 210, 235 and 163 at concentrations of 10, 25 and 50% (w/v) of the test substance in acetone/olive oil (4:1 v/v), respectively. No significant changes compared to the control value (group DPM = 176) were observed after treatment with the test substance. The stimulation indices were 1.2, 1.3 and 0.9 for concentrations of 10, 25 and 50%, respectively. Since there were no SI values greater than 3 when tested up to 50%, the test substance was considered not to be a skin sensitiser. The established EC3 value exceeds a concentration of 50%.

Based on the results of the available study on the skin sensitisation potential of the structural analogue Amides, C16-C18 (even), N,N'-ethylenebis, it may be concluded that Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis is not skin-sensitising, either.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Study not required according to Annex VII-X of Regulation (EC) No 1907/2006.

Justification for classification or non-classification

The available data on skin sensitisation of a substance structurally related to Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis according to Regulation (EC) No 1907/2006, Annex XI, 1.5 do not meet the criteria for classification according to Regulation (EC) No 1272/2008; therefore, Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis is not expected to exert a skin sensitisation potential, either, and the data are thus conclusive but not sufficient for classification.