Registration Dossier

- Skin irritation: not irritating in rabbits (one study acc. to OECD guideline 404 and EU method B.4, GLP), applied to the intact skin for 4 hours. Exposure to the test substance resulted in very slight or well defined erythema (score 1 - 2) and very slight or slight edema (score 1 - 2) in the treated skin areas of the three rabbits. Scaliness was seen in two animals after 7 days. The skin irritation resolved within 7 days after exposure in one animal and after 14 days in the other two animals.

- Eye irritation: irreversible effects to teh eye in rabbits (one study acc. to OECD guideline 405 and EU method B.5, GLP); undiluted test material applied to right eye of one animal. The eye lesions consisted of an injury of the cornea (opacity maximum grade 2), iridial irritation grade 1 and severe effects on the conjunctivae (grey/white coloration indicating necrosis, redness, chemosis and bloody discharge). Fluorescein examination could not be performed due to the bloody discharge. Scoring of the cornea, iris and eyelids was not possible in vivo 24 hours after instillation due swelling and exudation. Due to the severity of effects in this animal, no further animals were treated. The severity of effects on the eye necessitated intermediate sacrifice of the animal for ethical reasons approximately 27 hours after dosing.

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-10-22 to 2012-11-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted 24 April 2002

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: at least 6 weeks
- Weight at study initiation: at least 1.0 kg
- Housing: individually in labelled cages with perforated floors and shelters
- Diet (e.g. ad libitum): Pelleted diet for rabbits ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2012-10-22 To: 2012-11-20

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g of the test item

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.7 mL

Duration of treatment / exposure:

first animal: initially 3 minutes on one flank, consecutively one hour and 4 hours (separate patches) on the other flank;
further two animals: 4 hours

Observation period:

7 days (animal one)
14 days (animals 2 and 3)

Number of animals:

3 (one sex)

Details on study design:

TEST SITE
- Area of exposure: 2x3 cm
- % coverage: no data
- Type of wrap if used: Micropore tape secured with Coban elastic bandage

REMOVAL OF TEST SUBSTANCE
- Washing (if done): treated skin was cleaned of residual test substance using water after removal of dressing
- Time after start of exposure: animal 1: 3 min/1 h / 4 h; animals 2 and 3: 4 h

SCORING SYSTEM: Draize scale (OECD guideline 404)

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

other: mean 24, 48, 72 h

Score:

1.6

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal: #2, #3

Time point:

other: mean 24, 48, 72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 15 days

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: mean 24, 48, 72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #2

Time point:

other: mean 24, 48, 72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 8 days

Irritation parameter:

edema score

Basis:

animal #3

Time point:

other: mean 24, 48, 72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 8 days

Irritant / corrosive response data:

No skin irritation was seen after an exposure period of 3 minutes or 1 h to 0.5 g of the test substance.
4 h exposure to the test substance resulted in very slight or well defined erythema (score 1 - 2) and very slight or slight edema (score 1 - 2) in the treated skin areas of the three rabbits. Scaliness was seen in two animals after 7 days. The skin irritation resolved within 7 days after exposure in one animal and after
14 days in the other two animals.

Other effects:

No staining of the treated skin by the test substance was observed and no remnants of the test substance were present.
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Table 1: INDIVIDUAL SKIN IRRITATION SCORES

Animal # 1	3 minutes treatment site			1-hour treatment site			4-hours treatment site
Time after bandage removal	Erythema	Oedema	comments	Erythema	Oedema	comments	Erythema	Oedema	comments
Immediately	0	0	-	0	0	-	0	1	-
1 hour	0	0	-	NS	NS	-	1	1	-
3 hours	NS	NS	-	0	0	-	NS	NS	-
4 hours	0	0	-	0	0	-	NS	NS	-
5 hours	0	0	-	NS	NS	-	NS	NS	-
24 hours	0	0	-	0	0	-	2	1	-
48 hours	0	0	-	0	0	-	2	1	-
72 hours	0	0	-	0	0	-	1	1	-
7 days	0	0	-	0	0	-	0	0	-

Comments:

NS. Not scored according to protocol.

l. Scaliness.

Animal #	2 (4-hours treatment)			3 (4-hours treatment)
Time after exposure	Erythema	Oedema	comments	Erythema	Oedema	comments
1 hour	1	1	-	1	1	-
24 hours	2	1	-	2	1	-
48 hours	2	2	-	2	1	-
72 hours	2	1	-	2	1	-
8 days	1	0	I	1	0	I
15 days	0	0	-	0	0	-

Table 2: MEAN VALUE IRRITATION SCORES AFTER 4 HOURS OF EXPOSURE

Animal #	Mean 24, 48 and 72 hrs
	Erythema	Oedema
1	1.6	1.0
2	2.0	1.3
3	2.0	1.0

Interpretation of results:

GHS criteria not met

Conclusions:

Judgment is based on erythema and edema calculated as mean scores for each animal following grading at 24, 48 and 72 hours after removal of the test material, in consideration of reversibility. The following mean scores were calculated: erythema 1.6/2.0/2.0 and edema 1.0/1.3/1.0. All effects were fully reversible within 7 days in one animal and after 14 days in the two other animals.

Executive summary:

In a primary dermal irritation study according to OECD Guideline 404, adopted 24 April 2002, and EU Method B.4, May 2008, 3 young adult New Zealand white rabbit of one sex were dermally exposed to 0.5 g of Stearic acid 3-(dimethylaminopropyl)amide (100% a.i) to 6 cm² skin surface. The first animal was subsequently exposed for 3 minutes, 1 and 4 hours. Two further animals were exposed for 4 hours. Animals then were observed for 7 (animal #1) or 14 days (animals #2, #3).

In animal 1, no signs of skin reactions were recorded after removing the patch applied for 3 minutes and 1 hour in the observations carried out after removing the patches. After the 4 hours exposure to the test item, very slight oedema (grade 1) was observed one hour after removing the patch. Skin reactions observed in this animal during the observations made 24 and 48 hours after removing the patch were slight erythema (grade 2) and very slight edema (grade 1). After 72 hours, there was very slight erythema and very slight edema (grade 1) that was fully reversible within 7 days.

In animals 2 and 3, very slight edema (grade 1) and very slight erythema (grade 1) were observed 1 h after patch removal. At the 24, 48 and 72 h observations slight erythema (grade 2) and very slight or slight edema (grade 1 - 2) were noted. Scaliness was seen in these two animals after 7 days. The skin irritation resolved within 15 days.

The means of the erythema and edema evaluations at 24, 48 and 72 hours for the three animals after a four-hour exposure were 1.6, 2 and 2 (erythema) and 1, 1.3 and 1 (edema).

In this study, Stearic acid 3-(dimethylaminopropyl)amide is classified as not irritating to rabbit skin according the criteria of CLP, EU GHS (Regulation (EC) No 1272/2008).

Endpoint conclusion:

no adverse effect observed (not irritating)

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012-09-13 to 2012-09-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted 24 April 2002

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: 11-13 weeks
- Weight at study initiation: 2.88 kg
- Housing: individually
- Diet (e.g. ad libitum): pelleted rabbit diet, app. 100 g/day, hay + wooden sticks
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24°C
- Humidity (%): 40-70%
- Air changes (per hr):app. 15/h
- Photoperiod (hrs dark / hrs light):12/12

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated contralateral eye served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 53.6 mg (approximately 0.1 mL)

Duration of treatment / exposure:

eyes were not rindes after treatment

Observation period (in vivo):

24 h

Number of animals or in vitro replicates:

1

Details on study design:

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

SCORING SYSTEM: Draine (OECD Guideline 405)

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

other: ca 27 h

Score:

2

Max. score:

4

Reversibility:

other: no further observation due to severity of effects

Remarks on result:

other: Animal sacrificed and eye scored following the 24 hours observation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

other: ca 27 h

Score:

0

Max. score:

2

Reversibility:

other: no further observation due to severity of effects

Remarks on result:

other: Animal sacrificed and eye scored following the 24 hours observation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

other: ca. 27 h

Score:

3

Max. score:

3

Reversibility:

other: no further observation due to severity of effects

Remarks on result:

other: Animal sacrificed and eye scored following the 24 hours observation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

other: 24 h

Score:

3

Max. score:

3

Reversibility:

other: no furtehr observation due to severity of effects

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

other: 24 h

Score:

4

Max. score:

4

Reversibility:

other: no further observation due to severity of effects

Irritant / corrosive response data:

Instillation of 53.6 mg of the test substance into an eye of one rabbit resulted in severe effects on the eyes, necessitating intermediate sacrifice of the animal for ethical reasons approximately 27 hours after dosing. The eye lesions consisted of an injury of the cornea (opacity maximum grade 2), iridial irritation grade 1 and severe effects on the conjunctivae (grey/white coloration indicating necrosis, redness, chemosis and bloody discharge). Fluorescein examination could not be performed due to the bloody discharge. Scoring of the cornea, iris and eyelids was not possible in vivo 24 hours after instillation due swelling and exudation. Due to the severity of effects in this animal, no further animals were treated.

Other effects:

No remnants were noted one and 24 hours after instillation. White remnants of the test substance were found ex vivo in the conjunctival sac of the eye following sacrifice, 27 hours after instillation.
No symptoms of systemic toxicity were observed in the animal during the test period.

	1 h	24 h	Ex vivo (Animal sacrificed and eye scored following the 24 hours observation)
Cornea, opacity	0	n.a.	2
Iris	1	n.a.	0
Conjunctival redness	2	3	3
Chemosis	4	4	n.a.
Conjunctival discharge	2	3	n.a.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

In this study, Stearic acid 3-(dimethylaminopropyl)amide has irreversible effects on the eyes (Category 1).

Executive summary:

In a primary eye irritation study according to OECD guideline 405, adopted 24 April 2002 and EU method B.5, May 2008, 53.6 mg (approximately 0.1 mL) of Stearic acid 3-(dimethylaminopropyl)amide was instilled into the conjunctival sac of an eye of a male young adult New Zealand White rabbit. The eye was not rinsed after application. The animal was observed for 24 h. Irritation was scored by the method of Draize.

Instillation of the test substance into the eye resulted in severe effects on the eyes, necessitating intermediate sacrifice of the animal for ethical reasons approximately 27 hours after dosing.

The eye lesions consisted of an injury of the cornea (opacity maximum grade 2), iridial irritation grade 1 and severe effects on the conjunctivae (grey/white coloration indicating necrosis, redness, chemosis and bloody discharge).

Fluorescein examination could not be performed due to the bloody discharge. Scoring of the cornea, iris and eyelids was not possible in vivo 24 hours after instillation due to swelling and exudation. Due to the severity of effects in this animal, no further animals were treated.

In this study, Stearic acid 3-(dimethylaminopropyl)amide has irreversible effects on the eyes (Category 1).

Endpoint conclusion:

adverse effect observed (irreversible damage)

Endpoint conclusion:

no study available

Skin irritation

In an in vitro dermal irritation study according to OECD guideline 439, adopted 22 July 2010 and EU method B.46, August 2009, Stearic acid 3-(dimethylaminopropyl)amide was applied in triplicate to EPISKIN tissues for 15 min. PBS was used as negative control, 5% SDS as positive control. After exposure the skin tissues were thoroughly rinsed to remove the test substance and transferred to fresh medium. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed.

The relative mean tissue viability obtained after 15 minutes treatment with the test substance compared to the negative control tissues was 108%. Since the mean relative tissue viability was above 50%, the test substance is considered to be non-irritant.

The positive control had a mean cell viability of 5% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 6%, indicating that the test system functioned properly.

In this in vitro skin irritation test Stearic acid 3-(dimethylaminopropyl)amide was not irritating.

Although the in vitro test alone would have been sufficient for classification, labelling and risk assessment and is considered to be scientifically valid for the prediction of irritant and non-irritant chemicals for Annex VII, and also Annex VIII, a confirmatory in vivo test has been performed.

This was due to the unexpectedly severe outcome of the in vivo eye irritation test: although the preceding in vitro BCOP assay showed no evidence of the test substance being corrosive, Stearic acid 3-(dimethylaminopropyl)amide produced irreversible effects to the eye in vivo (for details, see below). This result has created some doubt concerning the reliability of the in vitro skin irritation test for the test substance. Thus, to take no risk for human health, a subsequent in vivo skin irritation test has been conducted.

In a primary dermal irritation study according to OECD Guideline 404, adopted 24 April 2002, and EU Method B.4, May 2008, 3 young adult New Zealand white rabbit of one sex were dermally exposed to 0.5 g of Stearic acid 3-(dimethylaminopropyl)amide (100% a.i) to 6 cm² skin surface. The first animal was subsequently exposed for 3 minutes, 1 and 4 hours. Two further animals were exposed for 4 hours. Animals then were observed for 7 (animal #1) or 14 days (animals #2, #3).

In animal 1, no signs of skin reactions were recorded after removing the patch applied for 3 minutes and 1 hour in the observations carried out after removing the patches. After the 4 hours exposure to the test item, very slight oedema (grade 1) was observed one hour after removing the patch. Skin reactions observed in this animal during the observations made 24 and 48 hours after removing the patch were slight erythema (grade 2) and very slight edema (grade 1). After 72 hours, there was very slight erythema and very slight edema (grade 1) that was fully reversible within 7 days.

In animals 2 and 3, very slight edema (grade 1) and very slight erythema (grade 1) were observed 1 h after patch removal. At the 24, 48 and 72 h observations slight erythema (grade 2) and very slight or slight edema (grade 1 - 2) were noted. Scaliness was seen in these two animals after 7 days. The skin irritation resolved within 15 days.

The means of the erythema and edema evaluations at 24, 48 and 72 hours for the three animals after a four-hour exposure were 1.6, 2 and 2 (erythema) and 1, 1.3 and 1 (edema).

In this study, Stearic acid 3-(dimethylaminopropyl)amide is classified as not irritating to rabbit skin according the criteria of CLP, EU GHS (Regulation (EC) No 1272/2008).

Eye irritation

An in vitro study was performed initially to assess the corneal irritation and damage potential of Stearic acid 3-(dimethylaminopropyl)amide by means of the BCOP assay using fresh bovine corneas according to OECD guideline 437, adopted 7 September 2009 and EU method B.47, December 2010.

Since no workable suspension of the test substance in physiological saline could be obtained, the test substance was used as delivered by the sponsor and added pure on top of the corneas (308 to 311 mg test substance). Physiological saline was used as negative control, 20% (w/v) Imidazole in physiological saline as positive control.

The corneas were incubated with Stearic acid 3 -(dimethylaminopropyl)amide for 240±10 min at 32±1°C. The test was performed in triplicates. After rinsing the corneas at least 3 times, opacity and permeability were determined. The in vitro irritancy score (IVIS) were calculated as mean opacity value + (15 x mean OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant. The positive and negative controls induced the appropriate responses.

The corneas treated with Stearic acid 3 -(dimethylaminopropyl)amide showed opacity values ranging from 1 to 4 and permeability values ranging from 0.990 to 2.790. The corneas were covered with small spots after the 240 minutes of treatment. The in vitro irritancy scores ranged from 16 to 44. The mean in vitro irritancy score was 29 after 240 minutes of treatment with Stearic acid 3-(dimethylaminopropyl)amide.

Because the testing result (mean in vitro irritancy score of 29) was well below an IVIS of 55.1 additional testing had to be conducted for classification and labelling purposes in accordance with the accepted integrated testing strategy (ITS) for assessing the eye irritation potential of substances (REACH TGD R7a).

In a primary eye irritation study according to OECD guideline 405, adopted 24 April 2002 and EU method B.5, May 2008, 53.6 mg (approximately 0.1 mL) of Stearic acid 3-(dimethylaminopropyl)amide was instilled into the conjunctival sac of an eye of a male young adult New Zealand White rabbit. The eye was not rinsed after application. The animal was observed for 24 h. Irritation was scored by the method of Draize.

Instillation of the test substance into the eye resulted in severe effects on the eyes, necessitating intermediate sacrifice of the animal for ethical reasons approximately 27 hours after dosing.

The eye lesions consisted of an injury of the cornea (opacity maximum grade 2), iridial irritation grade 1 and severe effects on the conjunctivae (grey/white coloration indicating necrosis, redness, chemosis and bloody discharge).

Fluorescein examination could not be performed due to the bloody discharge. Scoring of the cornea, iris and eyelids was not possible in vivo 24 hours after instillation due to swelling and exudation. Due to the severity of effects in this animal, no further animals were treated.

In this study, Stearic acid 3-(dimethylaminopropyl)amide has irreversible effects on the eyes (Category 1) according the criteria of CLP, EU GHS (Regulation (EC) No 1272/2008).

Respiratory irritation

No data on the respiratory irritation of Stearic acid 3-(dimethylaminopropyl)amide are available.

There are no data gaps for the endpoint irritation/corrosion. No human information is available for this endpoint. However, there is no reason to believe that these results would not be applicable to humans.

Skin irritation

Based on reliable, adequate and relevant data for Stearic acid 3-(dimethylaminopropyl)amide the classification criteria regarding skin irritation outlined in regulation (EC) 1272/2008 or the former European directive on classification and labelling 67/548/EEC are not met.

Eye irritation

Based on reliable, adequate and relevant data, Stearic acid 3-(dimethylaminopropyl)amide has to be classified in Category 1, irreversible effects on the eye according to CLP, EU GHS (Regulation (EC) No 1272/2008) and is assigned the hazard statement H318 and the signal word “Danger”. According to the criteria set out in directive 67/548/EECStearic acid 3-(dimethylaminopropyl)amide is classified as “Risk of serious damage to eyes” and is therefore assigned the risk phrase R41.