Registration Dossier

Administrative data

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, pre-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, pre-GLP): grade 3 redness and lesions of the iris grade 2 were observed throughout the study period (72 hours), reversibility was not observed

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:
other: Guideline study, GLP
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
2002
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals 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 (e.g. ad libitum): pelleted diet, ad libitum
- Water (e.g. ad libitum): 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
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:
other: 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:
other: mean 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:
other: mean 24, 48, 72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
animal #2
Time point:
other: mean 24, 48, 72
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
other: mean 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:
other: mean 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 oedema and moderate swelling oedema were observed in two animals. After 24 hours one animal showed no erythema and no oedema. 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 oedema decreased from slight swelling after 24 hours to very slight swelling after 48 hours and 7 days after patch removal the oedema 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 oedema 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)
Remarks:
Migrated information Criteria used for interpretation of results: other: CLP, EU GHS (Regulation (EC) No 1272/2008)
Conclusions:
In this skin irritation test in rabbit, DOPA-Glycinate 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.
Executive summary:

The acute dermal irritation of DOPA-Glycinate (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, DOPA-Glycinate 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:
other: Guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
1984
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
1981
Deviations:
no
GLP compliance:
no
Remarks:
At the time of the study conduct, GLP was not compulsory. However, the study was conducted in accordance with the principles of GLP.
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 (e.g. ad libitum): pelleted rabbit diet, ad libitum
- Water (e.g. ad libitum): 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:
animal: 1-6
Time point:
other: mean 24, 48, 72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal: 1-6
Time point:
other: mean 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:
animal: 1-6
Time point:
other: mean 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:
other: mean 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:
animal: 2-5
Time point:
other: mean 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.


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)
Remarks:
Migrated information Criteria used for interpretation of results: other: CLP, EU GHS (Regulation (EC) No 1272/2008)
Conclusions:
In this eye irritation test in rabbit, DOPA-Glycinate (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, the test substance has to be classified as Category 1 (irreversible effects on the eye).
Executive summary:

The acute eye irritation of DOPA-Glycinate (20% aqueous solution, “product by process”) 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.

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 and subsequent regulations, DOPA-Glycinate (20% a.i.) requires classification as Category 1 (irreversible effects on the eye).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

Several tests for the skin irritation/corrosion potential are available for DOPA-Glycinate.

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 DOPA-Glycinate 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 oedema (grade 4). These effects were completely reversible within 21 days after treatment. Thus, DOPA-Glycinate (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 DOPA-Glycinate (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 DOPA-Glycinate (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, 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, DOPA-Glycinate 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. According to the criteria set out in directive 67/548/EEC, DOPA-Glycinate is classified as corrosive, and assigned R34 “causes burns”.

 

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 DOPA-Glycinate (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) 1272/2008, DOPA-Glycinate has to be classified in Category 1, irreversible effects on the eye. According to the criteria set out in directive 67/548/EEC, DOPA-Glycinate (20% aqueous solution) is classified as R41 “Risk of serious damage to eyes”.

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 selection of skin irritation / corrosion endpoint:
OECD and EC guideline study, no deviations, GLP

Justification for selection of eye irritation endpoint:
OECD and EC guideline study, no deviations, pre-GLP (however, the study was conducted in accordance with the principles of GLP)

Effects on skin irritation/corrosion: corrosive

Effects on eye irritation: corrosive

Effects on respiratory irritation: irritating

Justification for classification or non-classification

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

According to the criteria set out in directive 67/548/EEC DOPA-Glycinate is classified as corrosive, and assigned R34 “causes burns”.

The technical material as manufactured is a liquid with ca. 20% w/w active matter 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. Based on the available reliable, adequate and relevant data with the material as manufactured, a specific concentration limit is set at 20%.

The 20% aqueous solution of DOPA-Glycinate 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) 1272/2008.

According to the former European directive on classification and labelling 67/548/EEC, the 20% aqueous solution of DOPA-Glycinate is classified as R38 (irritating to skin) and R41 (Risk of serious damage to eyes).