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

Endpoint summary

Description of key information

Skin irritation (OECD 404): not irritating, based on read-across from Amides, C16-C18 (even), N,N’-ethylenebis
Eye irritation (OECD 405): not irritating, based on read-across from Amides, C16-C18 (even), N,N’-ethylenebis

Respiratory tract irritation: not irritating, based on read-across from Amides, C16-C18 (even), N,N’-ethylenebis

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions. Occlusive dressing according to former guideline, prolonged exposure time (24 h), test substance was not removed after exposure.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

Occlusive dressing according to former guideline, prolonged exposure time (24 h), test substance was not removed after exposure.

GLP compliance:

no

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: own breeding
- Weight at study initiation: 2.16-2.45 kg
- Housing: separated in single cages
- Diet: standard diet ERKA 8300 (Robert Koch oHG, Hamm), ad libitum
- Water: ad libitum

Type of coverage:

occlusive

Preparation of test site:

other: one half shaved and the other additionally abraded

Vehicle:

other: polyethylene glycol 400

Controls:

not specified

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g, moistened with 1.2 mL polyethylene glycol 400

VEHICLE
- Amount(s) applied (volume or weight with unit): 1.2 mL

Duration of treatment / exposure:

24 h

Observation period:

72 h
Reading time points: 24, 48 and 72 h

Number of animals:

6

Details on study design:

TEST SITE
- Area of exposure: 2.5 x 2.5 cm on the shaved flank
- Type of wrap if used: the moistened test material was applied to 2.5 x 2.5 cm cellulose pads from Hansamed, the pads were fixed to the prepared application site and covered with an indifferent, impermeable polyethylene film (6-8 cm width). Subsequently, the whole trunk of the animals was wrapped with an elastic polyurethan warp bandage (permanent bandage K, Lohmann) and left in place for 24 hours.

REMOVAL OF TEST SUBSTANCE
- Washing: no

SCORING SYSTEM: Draize scoring system

Irritation parameter:

erythema score

Basis:

mean

Remarks:

of 6 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: intact skin

Irritation parameter:

edema score

Basis:

mean

Remarks:

of 6 animals

Time point:

24/48/72 h

Score:

0.11

Max. score:

4

Reversibility:

fully reversible within: 48 h in the 2 affected animals

Remarks on result:

other: intact skin

Irritant / corrosive response data:

No erythema was observed in abraded skin. Edema of grade 1 was observed in 4/6 animals in abraded skin after 24 hours which was fully reversible after 48 hours. After 24-h exposure to intact skin, animals #2 and #3 showed a mean edema score of 0.33 after 24, 48 and 72 h.

Other effects:

No further local and systemic effects were observed.

Interpretation of results:

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

Conclusions:

CLP: not classified

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Does not meet criteria of today standard methods. Solid substance not moistened, occlusive dressing according to former guideline, prolonged exposure time (24 h), test substance was not removed after exposure. No 48 h reading performed, insufficient duration of observation period and limited details on test animals and conditions.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

solid substance not moistened, occlusive dressing according to former guideline, prolonged exposure time (24 h), test substance was not removed after exposure; no 48 h reading performed, insufficient duration of observation period

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: adult, not further specified

Type of coverage:

occlusive

Preparation of test site:

other: shaved and abraded, respectively

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.5 mg

Duration of treatment / exposure:

24 h

Observation period:

72 h
Reading time points: 24 and 72 h

Number of animals:

12 (6 non-abraded, 6 abraded)

Details on study design:

TEST SITE
- Area of exposure: shaved or shaved and abraded back, respectively
- Type of wrap if used: Surgical patch of 1 inch x 1 inch, fixed to the application site by means of adhesive tape. The entire trunk of the rabbits was wrapped with an impervious material to maintain the test patches in position. Six rabbits were treated on the intact skin, the other six on the abraded skin. Abrasions were minor incisions through the stratum corneum, but not sufficiently deep to disturb the derma or to produce bleeding.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

SCORING SYSTEM: Draize

Irritation parameter:

erythema score

Basis:

mean

Remarks:

of 6 animals

Time point:

24/48/72 h

Score:

0.5

Max. score:

4

Reversibility:

not fully reversible within: 72 h (animal #4)

Remarks on result:

other: intact skin; no 48 h data are available; for calculation of mean scores, the 48 h values were assumed to be the same as those at 24 h (worst case assumption: persistence of effects seen at 24 h over 48 h)

Irritation parameter:

edema score

Basis:

mean

Remarks:

of 6 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: intact skin; no 48 h data are available; for calculation of mean scores, the 48 h values were assumed to be the same as those at 24 h (worst case assumption: persistence of effects seen at 24 h over 48 h)

Irritant / corrosive response data:

Irritants effects observed in abraded skin were even less pronounced and fully reversible within 72 hours. Although observed erythema was not fully reversible within the observation period of 72 hours in 1/6 animals after application to intact skin, the slight characteristics of the remaining effect and the full reversibility observed under more stringent experimental conditions in abraded skin make the assumption of full reversibility within a slightly longer period of time reasonable.

Other effects:

No further local or systemic effects were observed.

Table 1. Results of skin irritation study (intact skin)

Observation time	Rabbit no.
	1		2		3		4		5		6
	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema
24 h	0	0	1	0	1	0	1	0	0	0	1	0
48 h	No experimental data available; for calculation of mean scores, the 48 h values were assumed to be the same as those at 24 h (worst case assumption)
72 h	0	0	0	0	0	0	1	0	0	0	0	0

Table 2. Calculation of mean scores (intact skin)

	Rabbit no.
	1		2		3		4		5		6
	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema
Mean value 24 + 48 + 72 h*	0	0	0.67	0	0.67	0	1	0	0	0	0.67	0

*No 48 h data are available: for calculation of mean scores, the 48 h values were assumed to be the same as those at 24 h (worst case assumption: persistence of effects seen at 24 h over 48 h).

Interpretation of results:

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

Conclusions:

CLP: not classified

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Does not meet criteria of today standard methods. Solid substance not moistened, occlusive dressing according to former guideline, prolonged exposure time (24 h), test substance was not removed after exposure. No 48 h reading performed and limited details on test animals and conditions.

Qualifier:

according to guideline

Guideline:

other: Federal Regulations 16 CFR 1500.41: Method of testing primary irritant substances

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

solid substance not moistened, occlusive dressing according to former guideline, prolonged exposure time (24 h), test substance was not removed after exposure. No 48 h reading performed and limited details on test animals and conditions

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2.80-3.70 kg

Type of coverage:

occlusive

Preparation of test site:

other: shaved and abraded, respectively

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.5 g

Duration of treatment / exposure:

24 h

Observation period:

24 and 72 hours

Number of animals:

6

Details on study design:

TEST SITE
- Area of exposure: back and sides, closely clipped with an electric clipper, two sites per rabbit, each site 1 inch square in area. A site to the left of the spinal cord was abraded, while a site right to the spinal cord was left intact. The abrasions were sufficiently deep as to penetrate the stratum corneum, but not so deep as to disturb the derma or produce bleeding.
- Type of wrap if used: the test material was applied beneath surgical gauze square 1 inch x 1 inch, eight single layers thick, placed directly on the test site and secured with Dermicel tape. The animals were then wrapped with plastic sheeting secured with masking tape. After 24 hours, the sheeting and gauze patches were removed.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

SCORING SYSTEM: Draize scoring system

Irritation parameter:

erythema score

Basis:

mean

Remarks:

of 6 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: intact skin; no 48 h data are available; for calculation of mean scores, the 48 h values were assumed to be the same as those at 24 h (worst case assumption: persistence of effects seen at 24 h over 48 h)

Irritation parameter:

edema score

Basis:

mean

Remarks:

of 6 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: intact skin; no 48 h data are available; for calculation of mean scores, the 48 h values were assumed to be the same as those at 24 h (worst case assumption: persistence of effects seen at 24 h over 48 h)

Irritant / corrosive response data:

No effects were observed after exposure to the test substance on intact and abraded skin, respectively.

Other effects:

No further local or systemic effects were observed.

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 irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

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: Comparable to guideline study with acceptable restrictions. Only 72 h observation time, no informations on full reversibility.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

only 72h observation time, no informations on full reversibility

GLP compliance:

no

Species:

rabbit

Strain:

Himalayan

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Own breeding
- Weight at study initiation: 2.02-2.59 kg
- Housing: separated in single cages
- Diet: standard diet ERKA 8300 (Robert Koch oHG, Hamm), ad libitum
- Water: ad libitum

Vehicle:

other: polyethylene glycol 400

Controls:

other: right eye served as untreated control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 100 mg, moistened with 0.4 mL vehicle

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.4 ml

Duration of treatment / exposure:

24 h

Observation period (in vivo):

24 h
Reading time points: 1, 7, 24, 48 and 72 h

Number of animals or in vitro replicates:

6

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: physiol. saline
- Time after start of exposure: 24 h

SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: hand lens, additionally at 48 and 72 h time points: one drop of fluorescein 0.01%

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

of 6 animals

Time point:

24/48/72 h

Score:

0.17

Max. score:

4

Reversibility:

not fully reversible within: 72 h

Remarks on result:

other: observation period not sufficient to assess full reversibility

Irritation parameter:

iris score

Basis:

mean

Remarks:

of 6 animals

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: reversibility: not applicable

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

of 6 animals

Time point:

24/48/72 h

Score:

0.72

Max. score:

3

Reversibility:

not fully reversible within: 72 h

Remarks on result:

other: observation period not sufficient to assess full reversibility

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

of 6 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility: not applicable

Irritant / corrosive response data:

Although there is slight redness and corneal opacity left in 1/6 animals after 72 hours, the decrease of the scores over the observation time and the slight characteristics of the observations make it reasonable to anticipate full reversibility after a slightly longer observation period.

Table 1: Eye irritation scores after 1 and 7 h

	1 h						7 h
Rabbit No.	1	2	3	4	5	6	1	2	3	4	5	6
Cornea score	0	1	0	0	0	0	0	0	1	0	0	0
Iris score	0	0	0	0	0	0	0	0	0	0	0	0
Conjunctiva score	2	2	2	2	2	2	2	2	2	2	2	1
Chemosis score	2	2	2	2	2	2	1	1	1	1	1	1
Discharge score	3	3	3	3	2	3	2	1	1	1	0	1

Table 2: Eye irritation scores after 24, 48 and 72 h

	24 h						48 h						72 h
Rabbit No.	1	2	3	4	5	6	1	2	3	4	5	6	1	2	3	4	5	6
Cornea score	0	0	1	0	0	0	0	0	1	0	0	0	0	0	1	0	0	0
Iris score	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Conjunctiva score	1	1	1	2	2	0	1	1	1	1	1	0	0	0	1	0	0	0
Chemosis score	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Discharge score	1	1	2	1	0	1	0	0	0	0	0	0	0	0	0	0	0	0

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 irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

There is no data available on the skin and eye irritation 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 VIII, 8.1 and 8.2, 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 technical dossier (see IUCLID Sections 7.1 and 13).

Skin irritation

No study investigating the skin irritation potential of Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis is available. However, there exist four studies on the skin irritation 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.

In a study similar to OECD guideline 404, the test substance (0.5 g), moistened with 1.2 mL polyethylene glycol 400, was applied to intact or abraded skin of 6 rabbits (Leist, 1981). According to the former guideline, the test was performed under occlusive conditions, and the substance was not removed by washing. The scoring of skin reactions (erythema and edema) was performed 1, 24, 48 and 72 h after test substance application. The only observed effect on intact skin was a slight edema (grade 1) in 2/6 animals at the 24 h reading, which was fully reversible after 48 h. No erythema was observed on the intact skin of any treated animal. Although not relevant for classification, comparable effects of the test substance on abraded skin were noted (no erythema and slight edema in 4/6 animals, which reversed within 48 hours). The mean erythema and edema scores observed at 24, 48 and 72 h after exposure to intact skin were 0 and 0.11 for all 6 animals, respectively.

There is evidence from a further study to show that the test substance is not irritating to skin (Lina, 1981). Similar to the results of the first study, no edema and only slight erythema (grade 1) in 4/6 animals were observed on the intact skin after 24-h exposure to the test substance. At the end of the 72-h observation period, skin erythema was not fully reversible in 1/6 animals. However, this study does not meet the criteria of today's standards, since exposure to the test substance was longer than 4 h, only two readings were performed at 24 and 72 h and the observation period of 72 h was insufficient to assess the reversibility of effects. In contrast, the erythema observed on abraded skin was fully reversible within 72 h in 4/6 animals (grade 1). Although erythema after application to intact skin was not fully reversible within the observation period of 72 h, the slight characteristics of the remaining effect (grade 1, only 1/6 animals) and the full reversibility observed under more stringent experimental conditions in abraded skin make the assumption of full reversibility within a slightly longer period of time reasonable. Therefore, the test substance was not considered to be irritating to the skin in this study, either.

In the third available study, no effects were observed on the intact skin of 6 treated animals, and the same observation was made on abraded skin (Rinehart, 1978). Therefore, this study further supports the overall view that the substance is not irritating to the skin.

The fourth study was performed with only one animal (Lilja, 1981). Although not demonstrating any irritating effects, this study was not acceptable for assessment, as the animal was only exposed for 2 h and the only relevant reading of effects was done at 24 h.

Based on the available data on skin irritation 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 irritating to skin, either.

Eye irritation

No study investigating the eye irritation potential of Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis is available. However, there exist three studies on the eye irritation 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.

In the key study, 0.1 g of test substance, moistened with 0.4 mL of polyethyleneglycol 400, was applied to the left conjunctival sac of 6 rabbits (Leist, 1981). The right eyes remained untreated and served as controls. After 24 h, the eyes were washed with physiological saline and irritation reactions were assessed 24, 48 and 72 h after application. Corneal damage was additionally confirmed by application of 0.01% fluorescein at the 48- and 72 h readings. No iritis or chemosis were observed. Slight corneal opacity (grade 1) was noted in 1/6 animals, which was not fully reversible at the 72 h reading. Slight to moderate erythema (grade 1 and 2) was observed in 5/6 animals after 24 and 48 h, which declined at 48 h and fully reversed in 4 of the 5 concerned animals within 72 h. Slight erythema remained in 1/6 animals at the 72 h reading, which was the same animal also showing irreversible effects on cornea at this time point.

However, although there is slight redness and corneal opacity left in 1/6 animals after 72 h, which represents the end of the observation period, the decrease of the scores over the observation time and the slight characteristics of the observations make it reasonable to anticipate full reversibility after a slightly longer observation period. Therefore, the test substance was not considered to be irritating to the eyes.

Additional information on reversibility is available from two further studies (Lilja, 1981; Lilja, 1983), which were performed for screening purposes with only one animal per testing procedure. In both studies, the test substance remained in the eye without washing for an observation period of 7 days. Although some irritant effects on the iris and the conjunctiva were observed in both studies, these were reversible within a maximum of 7 days. No effects on cornea were observed in treated animals of both studies. However, the results of this study were not acceptable for risk assessment, as they were obtained from one animal only, and confirmation from further animals was lacking.

Based on the results of the studies on the eye irritation potential of the structural analogue Amides, C16-C18 (even), N,N'-ethylenebis, there is strong evidence that Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis is not irritating to the eye, either.

Respiratory tract irritation

No study investigating the acute or repeated dose toxicity via the inhalation route of Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis is available. However, there exist two studies on the acute inhalation toxicity of the structurally related substance Amides, C16-C18 (even), N,N'-ethylenebis. Therefore, read-across based on the analogue approach is performed.

In the GLP-study performed according to OECD guideline 403, 10 rats (5 per sex) were exposed whole body to a fixed gravimetrically determined concentration of 6.3 mg/L for 4 h (Siglin, 1987). The mass median aerodynamic diameter of the generated particles was determined to be 5.2 ± 1.7 µm, which is acceptable considering the technical standards and the requirements of the guideline at the time of the study. No mortality occurred up to the end of the 14-day observation period. Therefore, the LC50 was determined to be greater than 6.3 mg/L. Clinical signs indicative of transient respiratory irritation observed in this study comprised laboured breathing and/or rales and dark material around nose or mouth.

In a further study, 10 rats were exposed to a nominal exposure concentration of 58.2 mg/L for 1 hour only (Rusch, 1979). No mortality occurred under the conditions of this study. During exposure, no treatment-related abnormalities were observed. However, signs of a reversible irritant effect, as observed by mucoid and red nasal discharge as well as dry rales, were reported during the 14-day observation period.

The read-across substance Amides, C16-C18 (even), N,N'-ethylenebis is not classified for irritating properties and no signs of local irritation were observed when applied via the dermal route. The substance is not eye irritating. In the Acute Inhalation Toxicity studies transient and reversible respiratory tract irritation was observed in rats after exposure to an aerosol of the substance above the limit concentration for classification for acute toxicity. The substance is a solid with a low vapour pressure (0.0297 Pa at 25 °C). Therefore, based on the available data, classification according to CLP Regulation as STOT SE 3 (respiratory irritation) for Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis is not warranted.

Justification for classification or non-classification

The available data on skin, eye and respiratory tract irritation 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 potential for skin, eye and respiratory tract irritation, either, and the data are thus conclusive but not sufficient for classification.