Registration Dossier

Administrative data

Description of key information

For skin irritation, read across data were used from the test item 'Butanedioic acid, 2(or 3)-sulfo-, 4-[2-

[(1-oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts'. The

test item containing 39.8% active ingredient was predicted to be irritant to human skin based on the

three-dimensional EST 1000 human skin model (20 minutes exposure, followed by 42 hours incubation):

the mean cell viability was 1.3% of the negative controls, which was below the 50% cut-off value. It was

negative for corrosion in the same test model (3 minutes and 1 hour exposure): mean cell viability

values were 101.0% and 78.0%, respectively, which were above cut-off values for corrosion. A 5 and

10% solution with registered substance were previously tested to be non-irritant based on human

application to arm skin for 48 hours.
For eye irritation, the test substance containing 39.90% active ingredient was tested and predicted to be

a moderate irritant to human eye based on the in vitro HET-CAM model: the irritation score (IS) was 8.1,

so it was predicted to be a moderate irritant. In the in vivo eye irritation in rabbits , the same test item was

non-irritating, therefore not classified. Mean 24-72h scores were 0.11/4, 0.55/3 and 0.22/4 for cornea

opacity, conjuctivae redness and chemosis, respectively. A 10% solution of registered substance was

previously tested to be mildly irritant based on the Draize method in rabbit eyes, however reversible and

not classified.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
see attached read-across justification
Reason / purpose for cross-reference:
read-across source
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of 3 replicates; % versus negative control group
Run / experiment:
test group
Value:
1.3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Time point: 20 minutes, followed by 42h incubation.
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of 3 replicates; % versus negative control group
Run / experiment:
positive control group
Value:
1.3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Time point: 20 minutes, followed by 42h incubation.
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: No interactions of the test item with MTT were noted.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The viability of cells treated with the positive reference item, 5% SDS, was 1.3% of the negative controls and below the 50% cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.
All quality criteria required were fulfilled.
No interactions of the test item with MTT were noted.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control (NC): For each assay using valid batches, tissues treated with the NC exhibit OD reflecting the quality of the tissue that followed all shipment and receipt steps and all the irritation protocol process. The OD values of controls should not be below historical established lower boundaries.
- Acceptance criteria met for positive control (PC): Tissues treated with the PC, i.e. 5% aqueous SDS, should reflect the sensitivity retained by tissues and their ability to respond to an irritant substance in the conditions of each individual assay (e.g. viability ≤ 50% for the validated method)
- Acceptance criteria met for variability between replicate measurements: Associated and appropriate measures of variability between tissue replicates are defined (e.g. if standard deviations should be ≤ 18%).

The cell viability was measured by determining the optical density (OD) at a wavelength of 540 nm. An exposure time of 20 minutes was employed.

The test item, Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts,was applied to the model skin surface. Water for injection was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item.

The mean viability of cells exposed to the test item was 1.3% of the negative controls and, hence, below the 50% cut-off value.The test item was considered to be cytotoxic and is predicted to be irritant to skin in accordance with UN GHS category 2.

The viability of cells treated with the positive reference item, 5% SDS,was 1.3% of the negative controls and below the 50% cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the present test conditions read-across test item Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts, tested at an exposure time of 20 minutes, was cytotoxic and, hence, irritant to skin in an experiment employing an artificial three-dimensional model of human skin.

Executive summary:

The purpose of this study was to determine cytotoxic properties to skin cells, which might lead to irritation by read-across test item Butandioic acid, 2(or 3)-sulfo, 4 - [2 - [(1 -oxo(C12 -C18(even numbered) and C18 unsaturated)alkyl)amino]ethyl], esters, disodium salts to human skin, in an experiment with an artificial three-dimensional model of human skin. The EST1000 model was employed. The read-across test item containing 39.8% active ingredient was applied. The cell viability was measured by determining the optical density (OD) at a wavelength of 540 nm. An exposure time of 20 minutes was employed, followed by refreshment of the medium and further 42 hours incubation. Water for injection was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item. The mean viability of cells exposed to the read-across test item was 1.3% of the negative controls and, hence, below the 50% cut-off value. The read-across test item was considered to be cytotoxic and is predicted to be irritant to skin in accordance with UN GHS category 2.

The viability of cells treated with the positive reference item, 5% SDS, was 1.3% of the negative controls and below the 50% cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
see attached read-across justification
Reason / purpose for cross-reference:
read-across source
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean; % versus negative control group.
Run / experiment:
test group 3 minutes exposure
Value:
101
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean; % versus negative control group.
Run / experiment:
test group 1 hour exposure
Value:
78
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean; % versus negative control group.
Run / experiment:
positive control group 3 minutes exposure
Value:
4.1
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean; % versus negative control group.
Run / experiment:
positive control group 1 hour exposure
Value:
3.6
Negative controls validity:
valid
Positive controls validity:
valid

The read-across test item, Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts,was applied to the skin surface. Water for injection was used as the negative control. 8 N KOH was used as the positive reference item. Two exposure times of 3 minutes or 1 hour were employed.

In comparison to the negative controls, the mean viability of cells exposed to the test item was 101.0% after a 3-minute exposure period and 78.0% after a 1-hour exposure. The OD540 values were well above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of <50% or <15% for a 3-minute or 1-hour treatment, respectively. Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.

The mean viability of cells treated with the positive reference item 8 N KOH were 4.1% (3-minute incubation) and 3.6% (1-hour incubation) of the negative controls and were below the cut-off values and well within the range of historical background data of 3.3 – 47.4% (3-minute incubation) and 0.2 – 4.9% (1-hour incubation) of the negative controls (the last 6 experiments, not audited by the QAU-department). Hence, 8 N KOH caused pronounced corrosion in this skin model and is predicted to be corrosive to human skin.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the present test conditions read-across test item Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts tested at two exposure times of 3 minutes or 1 hour was non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin.
Executive summary:

The purpose of this study was to determine cytotoxic properties to skin cells which might lead to corrosion by read-across test item Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts to human skin, in an experiment with an artificial three-dimensional model of human skin. The EST-1000 model was employed. The test item containing 39.8% active ingredient was applied to the skin surface. Water for injection was used as the negative control. 8 N KOH was used as the positive reference item.Two exposure times of 3 minutes or 1 hour were employed. In comparison to the negative controls, the mean viability of cells exposed to the read-across test item was 101.0% after a 3-minute exposure period and 78.0% after a 1 -hour exposure.The OD540 values were well above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of <50% or <15% for a 3-minute or 1-hour treatment, respectively. Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.

The mean viability of cells treated with the positive reference item 8 N KOH were 4.1% (3 -minute incubation) and 3.6% (1 -hour incubation) of the negative controls and were below the cut-off values. Hence, 8 N KOH caused pronounced corrosion in this skin model and is predicted to be corrosive to human skin.

Endpoint:
skin irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
see attached read-across justification
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
read-across source
Number of animals:
4 volunteers per test substance
Irritation parameter:
overall irritation score
Basis:
other: individual scores
Time point:
other: 48 hours & 5 days
Score:
0
Irritant / corrosive response data:
No reactions were detected.

Table 1. Irritation

Subject

5% solution

10% solution

48 hours

5 days

48 hours

5 days

102

-

-

-

-

103

-

-

-

-

104

-

-

-

-

105

-

-

-

-

Result-No reaction detected

Interpretation of results:
not irritating
Remarks:
Criteria used for interpretation of results: expert judgment
Conclusions:
No reactions were detected with the read-across test item.
Executive summary:

A 1 cm2 of unmedicated white lint was soaked in the 5 and 10% read-across test item solution in comparison with toilet soaps, and applied to the skin of the left arm of 4 volunteers for 48 hours under waterproof adhesive dressing, followed by an observation period of 72 hours. No reactions were detected.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been conducted according to GLP and valid methods, therefore it is considered to be adequate, reliable and relevant for classifcation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
adopted October 02, 2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
Commision Regulation (EC) No. 440/2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
Himalayan
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxiclogy GmbH & Co. KG, Branch Löhndorf, 24601 Löhndorf/Post Wankendorf, Germany
- Age at dosing: Approx. 4.5 – 7.5 months
- Weight at dosing: Animal no. 1,2 and 3 : 2.7 kg each
- Housing: For 8 hours following test item application, the animals were kept singly in restrainers which allowed free movement of the head but prevented a complete body turn, wiping of the eyes with the paws and excluded irritation of the eye by excrements and urine. During the acclimatisation period and after the 8-hour period in restrainers, the animals were kept singly in cages with dimensions of 380 mm x 425 mm x 600mm (manufacturer: Dipl.Ing. W. EHRET GmbH, 16352 Schönwalde, Germany).
- Diet (e.g. ad libitum): Commercial diet , ssniff®K-H, ssniff Spezialdiäten GmbH, 59494 Soest, Germany, ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: At least 20 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C ± 3°C (maximum range)
- Humidity (%): 30% - 70% (maximum range)
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12 (about 150 lux at approx. 1.50 m room height)

IN-LIFE DATES: From: February 4, 2013 To: February 22, 2013
Vehicle:
unchanged (no vehicle)
Controls:
other: The left eye, which remained untreated, served as a control.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1mL
- Concentration (if solution): undiluted (39.90% solid content)
Duration of treatment / exposure:
Single instillation into the conjunctival sac
Observation period (in vivo):
Observations were performed at 1 hour, 24, 48, 72 hours, 7, 14, and 21 days
Number of animals or in vitro replicates:
3 males and 3 females
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, with physiol. saline
- Time after start of exposure: 24 h

SCORING SYSTEM:
The grades of ocular reaction (conjunctivae, cornea and iris) were recorded at each examination according to the following scheme:

Cornea
Opacity: degree of density (Readings taken from most dense area. The area of corneal opacity will be noted)
No ulceration or opacity: 0
Scattered or diffuse areas of opacity (other than slight dulling of normal lustre), details of iris clearly visible: 1
Easily discernible translucent area, details of iris slightly obscured: 2
Nacreous area, no details of iris visible, size of pupil barely discernible: 3
Opaque cornea, iris not discernible through the opacity: 4

Iris
Normal: 0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia, or injection, iris reacting to light (a sluggish reaction is considered to be an effect): 1
Haemorrhage, gross destruction, or no reaction to light: 2

Conjunctivae
Redness: (refers to palpebral and bulbar conjuntivae, excluding cornea and iris)
Normal: 0
Some blood vessels hyperaemic (injected): 1
Diffuse, crimson colour, individual vessels not easily discernible: 2
Diffuse beefy red: 3

Chemosis
Swelling (refers to lids and/or nictating membranes)
Normal: 0
Some swelling above normal: 1
Obvious swelling, with partial eversion of lids: 2
Swelling with lids about half closed: 3
Swelling, with lids more than half closed: 4


TOOL USED TO ASSESS SCORE:
slit lamp: prior to the administration and 1 hour, 24, 48 and 72 hours after the instillation
fluorescein: 24 hours after administration the eyes were treated additionally with fluorescein before being examined to aid evaluation of the cornea for possible lesions

Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0.11
Max. score:
4
Reversibility:
fully reversible within: 48 h
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 h
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
mean
Time point:
24/48/72 h
Score:
0.56
Max. score:
3
Reversibility:
fully reversible within: 72 h
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.67
Max. score:
3
Reversibility:
fully reversible within: 72 h
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 72 h
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.67
Max. score:
3
Reversibility:
fully reversible within: 72 h
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0.22
Max. score:
4
Reversibility:
fully reversible within: 48 h
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 h
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 h
Irritant / corrosive response data:
Under the present test conditions, a single instillation of 0.1 mL Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxododecyl)amino]ethyl]ester, disodium salt per animal into the conjunctival sac of the right eye of three rabbits caused following changes:
Corneal opacity (grade 1) was observed in animal no. one 24 hours after instillation.
Conjunctival redness was observed in all animals:
animal no. 1: 24 and 48 hours (grade 1) after instillation;
animal no. 2: 48 hours (grade 1) after instillation;
animal no. 3: 60 minutes (grade 2) and 24 and 48 hours (grade 1) after instillation.
Chemosis was observed in all animals:
animal no. 1: 60 minutes (grade 3) after instillation;
animal no. 2: 60 minutes (grade 2) and 24 hours (grade 1) after instillation;
animal no. 3: 24 hours (grade 1) after instillation.
The irises were not affected by instillation of the test item.
There were no systemic intolerance reactions.

Table 1. Examination of the treated eye

 

Animal no.: 1/2/3

Time after administration

Cornea opacity

Iris

Conjunctivae

Redness#

Chemosis##

Before dosing

0/0/0

0/0/0

0/0/0

0/0/0

60 minutes

0/0/0

0/0/0

0/0/2

3/2/0

24 hours

1/0/0

0/0/0

1/0/1

0/1/1

48 hours

0/0/0

0/0/0

1/1/1

0/0/0

72 hours

0/0/0

0/0/0

0/0/0

0/0/0

# refers to palpebral and bulbar conjunctivae; excluding cornea and iris

##swelling: refers to lids and/or nictating membrane

24 hours fluorescein test: all animals: no pathological findings

Interpretation of results:
GHS criteria not met
Conclusions:
Not irritating

Executive summary:

Under the present test conditions, a single instillation of 0.1 mL test item, containing 39.90% active ingredient, per animal into the conjunctival sac of the right eye of three rabbits caused following changes: corneal opacity (grade 1) in on animal 24 hours after instillation, conjunctival redness (grade 1) in all animals, chemosis in all animals up to 24 hours after instillation (grade 1 -3). The irises were not affected by instillation of the test item. Mean 24-72h scores were 0.11/4, 0.55/3 and 0.22/4 for cornea opacity, conjuctivae redness and chemosis, respectively.

All scores were reversible within 72 hours and fell below classification criteria, therefore the test item was not irritant for the eyes.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

Some data were available for lower concentrations of the registered substance (5 and 10% solutions) but not for higher concentrations, therefore read across data from 'Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts' were used as weight-of-evidence. Justification for read across within the subgroup of N-containing sulphosuccinates (N2 subcategory) is documented in a separate document attached in Section 13.

 

- In an in vivo (human) study, a 1 cm2 of unmedicated white lint was soaked with registered substance in the 5 and 10% test item solution in comparison with toilet soaps, and applied to the skin of the left arm of 4 volunteers for 48 hours under waterproof adhesive dressing, followed by an observation period of 72 hours (Baker Research Laboratories limited, 1962a). No reactions were detected.

- In a first in vitro study, the corrosive properties of read across substance Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)­alkyl))­amino]ethyl]esters, disodium salts, were studied in an experiment with a three-dimensional EST-1000 human skin model (Flügge, 2013a). The test item, containing 39.8% active ingredient, was applied for exposure times of 3 minutes or 1 hour. The mean viability of cells exposed to the test item was 101.0% after a 3-minute exposure period and 78.0% after a 1-hour exposure. These values were well above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of <50% or <15% for a 3-minute or 1-hour treatment, respectively. Therefore, the test item was non-corrosive in this human skin model and is predicted to be non-corrosive to human skin.

- In a second in vitro study, the irritant properties of read across substance 'Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts' to skin cells were also studied in an experiment with a three-dimensional EST-100 human skin model (Flügge, 2013b). The test item (containing 39.8% active ingredient) was applied for an exposure time of 20 minutes, followed by refreshment of the medium and further 42 hours incubation. The mean viability of cells exposed to the test item was 1.3% of the negative controls and, hence, below the 50% cut-off value. The test item was considered to be cytotoxic and is predicted to be irritant to skin in accordance with UN GHS category 2. The viability of cells treated with the positive reference item, 5% SDS, was 1.3% of the negative controls and below the 50% cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.

- According to ECHA progress report 2010 (p. 32), it is accepted that in vitro methods for skin irritation represent a full replacement of the in vivo method in a tiered testing strategy and in conjunction with in vitro skin corrosivity tests, if necessary. A positive result in the human skin model for irritation does not need to be confirmed by additional testing and shall lead to classification.

- In conclusion, the registered substance was predicted to be non-corrosive but irritant to human skin based on the three-dimensional EST 1000 human skin model, therefore CLP category 2 classsifciation is proposed. However, a concentration limit for non-classification can be used for solutions of 10% active ingredient or less.

 

Eye irritation:

Some supporting data were already available for lower concentrations of the registered substance (5 and 10% solutions); for the higher concentrations further in vitro and in vivo studies were performed with the registered substance.

 

- In a supporting in vivo eye irritation study (Doyle & Conine, 1983a) 0.1 mL test item containing 5 % active ingredient was instilled into the conjunctival sac of the right and left eye of 3 New Zealand White rabbits. After 24 hours the left and right eye of no animal showed any cornea lesion or iritis. Conjunctival redness was observed in the left and right eyes of all animals with score 2 after 1 hour. It decreased in all animals, and was fully reversible in both eyes after 96 hours. A chemosis was exhibited in both eyes of all animals after 1 hour with scores 1 to 2 which decreased and was fully reversible after 24 hours. Discharge was observed after 1 hour with scores of 1 to 2 in one animal in both eyes, and the left or right eye, respectively of the two other animals. After 24 hours discharge was fully reversible in the left and right eye of all animals.

- In a second supporting in vivo eye irritation study (Doyle and Conine, 1983b) test item containing 10 % active ingredient was instilled into the conjunctival sac of the right and left eye of 3 New Zealand White rabbits. After 24 hours the left and right eye of one animal showed a cornea lesion (score 1) which were fully reversible after 48 hours. In no eye of any animal an iritis was observed. Conjunctival redness was observed in all eyes of all animals with score 2 to 3 until 24 hours. It decreased in all animals, but was not reversible in both eyes of one animal after 7 days. A chemosis was exhibited in all eyes of all animals after 1 hour with scores 2 to 3 which decreased and was fully reversible after 48 hours. Discharge was observed after 1 hour with scores of 2 to 3 in all animals, which also decreased. After 48 hours discharge was fully reversible. The observation time did not extend beyond 7 days, even though an eye response was still present in one animal at this time, therefore, the reversibility of the eye effects cannot be evaluated and the severity of the eye irritation potential of the test substance cannot be classified. However, in the course of the examinations a clear tendency of reversibility was seen and at the last observation only one animal showed conjunctival redness with score 1. Therefore a complete reversibility of the eye effects can be expected.

- The eye irritancy potential of the test item containing 39.9% active ingredient was tested in vitro by means of a supporting chorioallantoic membrane of hens' eggs (HET-CAM) (Haferkorn, 2013c). The eggs treated with the test item revealed an effect with an irritation score (IS) of 8.1, so it was predicted to be a moderate irritant. The positive control items 0.1 N NaOH or 1% SDS caused the expected effect with irritation scores of 18.5 or 10.0, respectively and, hence, were well within the historical data-range. No effects were observed in the negative control 0.9% NaCl solution.

- In a key in vivo study, a single instillation of 0.1 mL test item, containing 39.90% active ingredient, was put into the conjunctival sac of the right eye of three rabbits (Hansen, 2013a). This caused following changes: corneal opacity (grade 1) in one animal 24 hours after instillation, conjunctival redness (grade 1) in all animals, chemosis in all animals up to 24 hours after instillation (grade 1 -3). Mean 24-72h scores were 0.11/4, 0.55/3 and 0.22/4 for cornea opacity, conjunctivae redness and chemosis, respectively. The irises were not affected by instillation of the test item. All scores were reversible within 72 hours and fell below classification criteria, therefore the test item was not irritant for the eyes. This study overrules the in vitro prediction study for the tested concentration.

-In conclusion, the registered substance was predicted not to induce severe eye damage, but to induce severe eye irritation based on the three-dimensional EST 1000 human skin model, therefore CLP category 2 classification is proposed. However, a concentration limit for non-classification can be used for solutions of 39.90% active ingredient or less.

 

Conclusion:

- A subgroup CLP category 2 classification was decided for skin irritation, with concentration limit of 10% for non-classification of registered substance.

- A subgroup CLP category 2 classification was decided for eye irritation, with concentration limit of 40% for non-classification of registered substance.

-More information on the subgroup classification is provided in the read-across justification, separately attached in Section 13. 

 

Justification for selection of skin irritation / corrosion endpoint: Although the in vitro study for irritation was selected, the corrosion study was equally valuable in a weight-of-evidence approach. 

Justification for selection of eye irritation endpoint: Last study in weight of evidence approach. 

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

The registered substance is classified for skin irritation according to CLP regulation (No. 1272/2008 of 16 December 2008) as Category 2, with signal word 'warning' and hazard statement: H315 - Causes skin irritation. However, a concentration limit of 10% for non-classification can be applied.

For the eye, the test substance is classified according to CLP regulation as Category 2, with signal word 'warning' and hazard statement: H319 -Causes serious eye irritation. However, a concentration limit of 40% for non-classification can be applied.