Amines, N-C10-16-alkyltrimethylenedi-, reaction products with chloroacetic acid

Endpoint summary

Description of key information

Skin irritation:
- neat substance: corrosive in rabbits (OECD TG 404, GLP): pure active substance (99.4% a.i.) applied to the intact skin for 4 hours, 1/3 animals was noted with full thickness destruction of the skin tissue at the 48- and 72-hour reading, mild to severe and early-onset signs of irritation in 2/3 animals (reversible in these animals within 14 d)
- 20% aqueous solution: irritating in rabbits (OECD TG 404, GLP): substance applied to the intact skin for 4 hours, 3/3 animals showed grade 4 erythema and grade 4 edema (reversible within 21 d)

Eye irritation:
- neat substance corrosive based on results from skin irritation study;
- 20% aqueous solution: irreversible effects to the eye in rabbits (OECD TG 405, GLP): grade 3 redness and lesions of the iris grade 2 were observed throughout the study period (72 hours), reversibility was not observed

- 1% aqueous solution: reversible effects to the eye in rabbits (OECD TG 405, GLP): mean grade 2 redness and chemosis was observed after 24/48/72 hour observation period, reversibility was not observed until 72 hours

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elevage Scientific des Dombes, Chatillon sur Chalaronne, France
- Age at study initiation: 14–15 weeks (male), 12–13 weeks (females)
- Weight at study initiation: 2229 – 2309 g
- Housing: individually
- Diet: pelleted diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: not stated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g (lyophilised test substance)

Duration of treatment / exposure:

4 h

Observation period:

14 d

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 16 cm² (4.0 cm x 4.0 cm) on a flank

REMOVAL OF TEST SUBSTANCE
- Washing (if done): tap water
- Time after start of exposure: 4 h

SCORING SYSTEM: Draize scale

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24 h

Score:

4

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: score of this animal was not assessable after 48 and 72 hours due to deep necrosis

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

other: not expected to be reversible

Remarks on result:

other: this animal was killed for ethical reasons after the 72-hour reading due to the deep necrosis observed

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 d

Remarks on result:

other: scaling was observed on days 7 and 10 (reversible within 14 d)

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 7 d

Irritant / corrosive response data:

One hour after patch removal slight erythema, well-defined erythema, severe erythema were observed in all animals and slight swelling edema and moderate swelling edema were observed in two animals. After 24 hours one animal showed no erythema and no edema. The well-defined erythema persisted until 72 hours, decreased to slight erythema 7 days after patch removal and was gone on day 14. In animal no.9 the edema decreased from slight swelling after 24 hours to very slight swelling after 48 hours and 7 days after patch removal the edema was gone. The severe erythema of animal no.7 persisted until 24 hour reading; it was noted with dry skin from the 24- to the 72-hour reading and was not assessable due to deep necrosis at 48 and 72 hour evaluation. The edema decreased from moderate swelling after 24 hours to slight swelling after 48 hours and very slight swelling after 72 hours, then animal no. 7 was killed for ethical reasons.
The cutaneous reactions were reversible in one animal after 24 hours, in another animal within 14 days after removal of the patch. The third animal was sacrificed after the 72 hour reading for ethical reasons.
No staining produced by the test item of the treated skin was observed.

Other effects:

No clinical signs of systemic toxicity were observed in the animals during the study. One animal was sacrificed for ethical reasons after the 72-hour reading.
In one animal (no.9) on day 7 and day 10 scaling was present.
The body weights of all rabbits were considered to be within the normal range of variability.

 

animal	observation	erythema	edema	scaling
#1 #2 #3	1 h	4 1 2	3 0 2	0 0 0
#1 #2 #3	24 h	4 0 2	3 0 2	0 0 0
#1 #2 #3	48 h	n.a. 0 2	2 0 1	0 0 0
#1 #2 #3	72 h	n.a. 0 2	1 0 1	0 0 0
#1 #2 #3	7 d	-** 0 1	-** 0 0	-** 0 x
#1 #2 #3	10 d	-** 0 1	-** 0 0	-** 0 x
#1 #2 #3	14 d	-** 0 0	-** 0 0	-** 0 0

 

n.a. : score of this animal was not assessable after 48 and 72 hours due to deep necrosis

-**: this animal was killed for ethical reasons after the 72-hour reading due to the deep necrosis observed

x: scaling present

Interpretation of results:

Category 1C (corrosive) based on GHS criteria

Conclusions:

In this skin irritation test in rabbit, the registration substance caused full thickness destruction of the skin tissue at the application site in one animal. Thus, the test substance has to be classified as corrosive Category 1C according to the criteria laid down in Regulation (EC) No 1272/2008 as well as the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations.

Executive summary:

The acute dermal irritation of the registration substance (lyophilised, 99.4% a.i.) was tested in 3 New Zealand white rabbits, according to EC method B.4 (2004/73/EEC) and OECD guideline 404 (2002).Rabbits were applied a dose of 0.5 g on the shaved intact skin for a period of 4 hours (semi-occlusive).

The application of the test item to the skin resulted in mild to severe and early-onset signs of irritation such as erythema, oedema and scaling. These effects were reversible and were no longer evident in the two surviving animals 14 days after treatment, the end of the observation period for all animals. One animal was noted with dry skin from the 24- to the 72-hour reading as well as with deep necrosis (full thickness destruction of the skin tissue) at the 48- and 72-hour reading. Thereafter, it was sacrificed for ethical reasons.

According to the requirements specified by Regulation (EC) 1272/2008 and subsequent regulations, the registration substance requires classification for skin corrosion/irritation with classification category 1C, signal word “danger” and the hazard phrase “H314: Causes severe skin burns and eye damage” according to the classification criteria of Regulation 1272/2008.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

1981

Deviations:

yes

Remarks:

duration of observation was not extended in order to sufficiently evaluate fully the reversibility

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harald Schriever, Rabbit farm, Bremervörde, Germany
- Weight at study initiation: 2.4–2.6 kg
- Housing: individually
- Diet: pelleted rabbit diet, ad libitum
- Water: drinking water, ad libitum
- Acclimation period: at least 7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18±2
- Humidity (%): 50-85
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

other: The untreated right eyes served as controls.

Amount / concentration applied:

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

Duration of treatment / exposure:

Group 1: Animal 1–3; no removal of test substance
Group2: Animal 4–6 exposure for 4 sec.

Observation period (in vivo):

72 h

Number of animals or in vitro replicates:

6

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Washed out with 10 mL lukewarm water (animal 4–6).
- Time after start of exposure: 4 sec

SCORING SYSTEM: Draize scale

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animals 1-3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

iris score

Basis:

mean

Remarks:

animals 1-3

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not fully reversible within: 72 h

Remarks on result:

other: reversibility cannot be judged as the observation was not continued after the 72 h time point

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animals 1-3

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not fully reversible within: 72 h

Remarks on result:

other: reversibility cannot be judged as the observation was not continued after the 72 h time point

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

not fully reversible within: 72 h

Remarks on result:

other: reversibility cannot be judged as the observation was not continued after the 72 h time point

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animals 2, 3

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

not fully reversible within: 72 h

Remarks on result:

other: reversibility cannot be judged as the observation was not continued after the 72 h time point

Irritant / corrosive response data:

Reversibility of the eye reactions could not be observed within the 72 hour-study period. All animals showed absence of the light reflex (iridial response grade 2) which persisted for 72 hours. According to the guidelines, this lesion is generally not reversible.
According to most recent guideline the test substance should not be washed out. Thus, only results from animals 1 to 3 were evaluated where the test substance was not washed out.

Table A6.1.4 -4:Acute eye irritation in rabbits of group 1 (no removal of test substance).

	Cornea	Iris	Conjunctiva
Score (animal #1, #2, #3)	Opacity		Redness	Chemosis
1 h	0/0/0	2/2/2	3/3/3	3/4/4
24 h	0/0/0	2/2/2	3/3/3	3/4/4
48 h	0/0/0	2/2/2	3/3/3	3/4/4
72 h	0/0/0	2/2/2	3/3/3	3/4/4
Average 24, 48, 72 h	0/0/0	2/2/2	3/3/3	3/4/4

 

Table A6.1.4 -5:Acute eye irritation in rabbits of group 2 (removal of test substance).

	Cornea	Iris	Conjunctiva
Score (animal #4/#5/#6)	Opacity		Redness	Chemosis
1 h	0/0/0	2/2/2	2/3/3	3/4/4
24 h	0/0/0	2/2/2	3/3/3	4/4/4
48 h	0/0/0	2/2/2	3/3/3	4/4/4
72 h	0/0/0	2/2/2	3/3/3	4/4/4
Average 24, 48, 72 h	0/0/0	2/2/2	3/3/3	4/4/4

 

Interpretation of results:

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

Conclusions:

In this eye irritation test in rabbit, the registration substance (20% a.i.) caused diffuse beefy redness and swelling with lids about half closed or more than half closed (grade 3) as well as lesions of the iris grade 2. Thus, Amines, N-C10-16-alkyltrimethylenedi-, reaction products with chloroacetic acid is classified according to the criteria laid down in Regulation (EC) No 1272/2008 as well as the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations as Category 1 (irreversible effects on the eye).

Executive summary:

The acute eye irritation of the registration substance (20% aqueous solution) was tested in New Zealand White rabbits, according to EC method B.5 (1984) and OECD guideline 405 (1981). Six rabbits were administered a dose of 0.1 mL test substance into the left eye. In one group consisting of three animals the eyes were not washed, while in the other group the test substance was removed after 4 seconds. According to the most recent OECD guideline 405 test substance should not be washed out after treatment. Thus, only animals where the test substance was not washed out were considered for classification and labelling.

However, the results with and without removal of the test substance were consistent. No corneal opacity was observed during the study. Diffuse beefy redness and swelling with lids about half closed or more than half closed as well as lesions of the iris grade 2 (no reaction to light, haemorrhage and/or gross destruction) were observed throughout the study period. Reversibility was not observed within the observation period of 72 hours. According to the guidelines, absence of the light reflex (iridial response grade 2) which persists for 72 hours is generally not reversible.

Thus, according to the requirements specified Regulation (EC) 1272/2008 as well as Labelling of Chemicals (GHS) of the United Nations, Amines, N-C10-16-alkyltrimethylenedi-, reaction products with chloroacetic acid requires classification as Category 1 (irreversible effects on the eye).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

Several tests for the skin irritation/corrosion potential are available for Amines, N-C10-16-alkyltrimethylenedi-, reaction products with chloroacetic acid.

Anhydrous active substance is not produced by the regular manufacturing process; the active substance as manufactured is the 20% aqueous solution.

The 20% a.i. aqueous solution of registration substance was first tested in 3 New Zealand White rabbits, according to EC method B.4 (1984) and OECD guideline 404 (1981). 0.5 mL of the test substance was applied to the intact skin of rabbits under semi-occlusive conditions for 4 hours.

All three rabbits showed severe erythema (grade 4, including moderate ischemic necrosis, moderate incrustation) and severe edema (grade 4). These effects were completely reversible within 21 days after treatment. Thus, the registration substance (20% a.i.) is irritating to the skin.

 

As the results were on the borderline to corrosive, a further in vitro test with the neat substance was performed.

The skin corrosion of the lyophilised registration substance (100%) was tested in a human skin model in vitro, according to EC method B.40 (2000/33/EC) and OECD guideline 431 (2002). The cells were treated with a dose of 25.0-27.0 mg wetted test item.

After treatment, the absorbance values for the negative control were above the required acceptability criterion of OD ≥ 0.8 for both intervals. The viability of the positive control met the acceptance criterion with ≤30% concerning the 3 minute treatment (29.6%) and the 1 hour interval (6.12%). These criteria validate the test system.

After treatment with the test substance, the relative absorbance values were decreased to 90.6% for the 3 minute treatment but not to <50%. After 1 hour treatment the relative absorbance values were reduced to 55.0% but not to <15%. Therefore, the test item was considered to be non-corrosive in this in vitro test.

 

At the time of the conduct of the test, a further confirmatory test in vivo was required, thus a dermal irritation study according to OECD guideline 404 was performed with the lyophilised registration substance (99.4% a.i.) in 3 New Zealand white rabbits. The application of the test item to the skin resulted in mild to severe and early-onset signs of irritation such as erythema, edema and scaling. These effects were reversible and were no longer evident in the two surviving animals 14 days after treatment, the end of the observation period for all animals. One animal was noted with dry skin from the 24- to the 72-hour reading as well as with deep necrosis (full thickness destruction of the skin tissue) at the 48- and 72-hour reading. Thereafter, it was sacrificed for ethical reasons. According to the requirements specified by Regulation (EC) No 1272/2008 and GHS-UN, Amines, N-C10-16-alkyltrimethylenedi-, reaction products with chloroacetic acid requires classification for skin corrosion/irritation with classification category 1C, signal word “danger” and the hazard phrase “H314: Causes severe skin burns and eye damage”.

 

Eye irritation

In accordance with REACH-Regulation (Annex VIII, 8.2.1, column 2) testing of eye irritating properties of the neat substance in vivo does not need to be conducted because the substance is already classified for irreversible effects on the eye based on the outcome of the in vivo skin irritation study ("Causes severe skin burns and eye damage").

However, an in vivo eye irritation study is available with the registration substance (20% aqueous solution) which constitutes the active substance as manufactured.

Diffuse beefy redness and swelling with lids about half closed or more than half closed as well as lesions of the iris grade 2 (no reaction to light, haemorrhage and/or gross destruction) were observed throughout the study period of the eye irritation study. Reversibility was not observed within the observation period of 72 hours. According to the guidelines, absence of the light reflex (iridial response grade 2) which persists for 72 hours is generally not reversible.Thus, according to the requirements specified by regulation (EC) No 1272/2008 and GHS-UN, the registration substance has to be classified in Category 1, irreversible effects on the eye.

A further study according to OECD TG 405 is available with a 1% aqueous solution of the registration substance. Reversible effects on the iris were observed 24 hours after treament (grade 1 in one animal). Conjunctival redness and chemosis were observed at the 24/48/72 h observation with the following grades for animals 1 to 3: redness: 2, 1.67 and 2, respectively and chemosis: 2, 2 and 1.67, respectively. The observation period lasted only 72 hours. However, it can be assumed that the effect are reversible because the grades of conjunctival redness and chemosis decreased over the observation time. Thus, according to the requirements specified by regulation (EC) No 1272/2008 and GHS-UN, the 1% aqueous solution of the registration substance has to be classified in Category 2A , reversible effects on the eye.

Respiratory irritation

There are currently no validated animal or in vitro tests that deal specifically with respiratory tract irritation. The Guidance on the Application of the CLP Criteria, Version 4.0, Nov. 2013 states that: "In general, a classification for corrosivity is considered to implicitly cover the potential to cause RTI [= respiratory tract irritation] and so the additional [STOT-SE] Category 3 is considered to be superfluous".

The classification of the substance as corrosive, Category 1C is considered to cover and communicate the potential for respiratory tract irritation adequately. 

Justification for classification or non-classification

Based on reliable, adequate and relevant data, the registration substance has to be classified in Category 1C according to CLP, EU GHS (Regulation (EC) No 1272/2008) and GHS-UN and is assigned the hazard statement H314 “Causes severe skin burns and eye damage”.

The technical material as manufactured is a liquid with ca. 20% w/w active ingredient and ca. 80% w/w water. A water-free form of the technical material (dry technical material) is not manufactured or sold for commercial purposes.

The 20% aqueous solution of the registration substance is classified as irritating to the skin (Category 2) and causing irreversible effects on the eye (Category 1) and labelled with H315 (Causes skin irritation) and H318 (Causes serious eye damage) according to regulation (EC) No 1272/2008 and GHS-UN. A 1% aqueous solution of the registration substance is causing reversible effects on the eye (Category 2A) and has to be labelled with H319 (Causes serious eye irritation) according to regulation (EC) No 1272/2008 and GHS-UN.