Registration Dossier

Administrative data

Description of key information

While in vitro studies on skin corrosion and irritaiton did not indicate a skin irritating property of the test substance, the in vivo skin irritation test in rabbits gave a borderline positive result and the substance is classified as irritating to skin. In vitro eye irritation assays indicated that the substance should be regarded as a severe eye irritant. Further animal studies were therefore not performed.

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
Study period:
2011/06/07 to 2011/07/05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
Method B4 Acute Toxicity (Skin Irritation) EC No. 440/2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK
- Age at study initiation: 12 to 20 weeks old
- Weight at study initiation: 2.35 to 2.62 kg
- Housing: Individually housed in suspended cages
- Diet (e.g. ad libitum): 2930 Teklad Global Rabbit Diet ad libitum
- Water (e.g. ad libitum): Mains drinking water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 degrees C
- Humidity (%): 30 to 70%
- Air changes (per hr): At least 15 exchanges per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

IN-LIFE DATES: From: 2011/06/07 To: 2011/07/05
Type of coverage:
semiocclusive
Preparation of test site:
other: Clipped with veterinary clippers
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 ml
- Concentration (if solution): undiluted

Duration of treatment / exposure:
One animal initially treated: three suitable sites with test article applied. One patch removed at either 3 minutes, 1 hour or 4 hours after application. Any residual test article was removed by gentle swabbing with cotton wool soaked in distilled water.

After consideration of the reactions in the first animal, an additional two animals were treated with 0.5 ml of test item. One patch was applied to the back of each of the two animals and allowed to remain in contact for 4 hours.
Observation period:
Observations and scoring of the skin reactions were made immediately after removing the patch, at 1, 24, 48 and 72 hours. Additional observations were made on days 7 and 14 to assess the irreversibility of the lesions.

Body weights were recorded on the day of dosing and at the end of the observation period
Number of animals:
Three male New Zealand White rabbits
Details on study design:
TEST SITE
- Area of exposure: under 2.5 cm square cotton gauze patch.
- Type of wrap if used: Strip of surgical adhesive tape, with trunk wrapped in an elasticized corset.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Gentle washing with cotton wool soaked in distilled water.
- Time after start of exposure: Immediately after patch removal (3 minutes, 1 hour or 4 hours).

SCORING SYSTEM: Draize J H (1959)
Erythema and Eschar Formation Value
No erythema 0
Very slight erythema 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema to eschar formation
preventing grading of erythema 4

Oedema formation
No oedema 0
Very slight oedema 1
Well-defined oedema 2
Moderate to severe oedema 3
Severe oedema 4
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
other: 24 and 72 hours
Score:
ca. 4
Reversibility:
fully reversible within: 14 days
Remarks on result:
other: 4 hour exposure, all three animals
Irritation parameter:
erythema score
Basis:
animal #1
Remarks:
mean 24, 48, 72 h
Time point:
other: 24, 48, 72 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 14 days
Remarks on result:
other: 4 h exposure, light brown discoloration of the epidermis at 48 and 72 h, loss of skin elasticity and flexibility at 72 h, hardened dark coloured and undulating scab at 72 h and 7d.
Irritation parameter:
erythema score
Basis:
animal #2
Remarks:
mean 24, 48, 72 h
Time point:
other: 24, 48, 72 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 14 days
Remarks on result:
other: 4 h exposure, light brown discoloration of the epidermis at 48 and 72 h, moderate desquamation at day 7, no reading at day 14.
Irritation parameter:
erythema score
Basis:
animal #3
Remarks:
mean 24, 48, 72 h
Time point:
other: 24, 48, 72 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: 4 h exposure, light brown discoloration of the epidermis at 24, 48 and 72 h, hardened dark coloured scab at 24, 48 and 72 h, moderate desquamation on day 7, no reading on day 14.
Irritation parameter:
edema score
Basis:
animal #1
Remarks:
mean 24, 48, 72 h
Time point:
other: 24, 48, 72 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 14 days
Remarks on result:
other: 4 h exposure
Irritation parameter:
edema score
Basis:
animal #2
Remarks:
mean 24, 48, 72 h
Time point:
other: 24, 48, 72 h
Score:
1.67
Max. score:
2
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: 4 h exposure
Irritation parameter:
edema score
Basis:
animal #3
Remarks:
mean 24, 48, 72 h
Time point:
other: 24, 48, 72 h
Score:
2.33
Max. score:
3
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: 4 h exposure
Irritant / corrosive response data:
See Any other information on results section for data used in calculating primary irritation index.
Other effects:
All three animals showed expected body weight changes over the course of the study. Day 0 to Day 14 weight change in animal 1 was 0.23 kg. Animals 2 and 3 gained 0.08 and 0.17 kg over Days 0 to Day 7 of the study.
 Skin reaction  Observation time  1st rabbit  2nd rabbit  3rd rabbit  Total
 Eryethema/eschar  immediately  2  1  1  (4)
 Erythema/eschar  1 hour  2  1  1  (4)
 Erythema/eschar  24 hours  2  2  2StBr  6
 Erythema/Eschar  48 hours  2Br 2Br  2StBr  (6)
 Erythema/Eschar  72 hours  2BrLeLfStSw  2Br  2StBr  6
 Erythema/Eschar  7 days  ?eCfStSw  OD  OD  (0 -4)
 Erythema/Eschar  14 days  0  -  -  (0)
 ( ) = values not used in calculating PII Br = brown discoloration Le = loss of skin elasticity Lf = Loss of skin flexibility St = hardened dark brown scab Sw = Scab undulating Cf = crust formation D = moderate desquamation ?e = adverse reaction preventing evaluation of erythema ?od = adverse reaction preventing evaluation of edema - = not applicable

 

 
 

Skin reactions after 3 min and 1 hour of exposure (one male animal):

 
 Skin reaction observation time following patch removal Score 3 min exposure  Score 1 h exposure
Erythema/Escharformation  0 to 1 h  0  0
   24 h  0  1
   48 h  1  2
   72 h  2Bl  2Bl
   7 days  0D  ?eCf
   14 days  0G  0
       
 Edema formation  0 to 1 h  0  0
   24 h  0  0
   48 h  0  1
  72 h   2  2
   7 days  0  ?od
   14 days  0  0
 Bl: Blanching of the skinD: Moderate desquamationCF: crust formationG: glossy skin?e: Adverse reaction preventevaluation of erythema?od: Adverse reactions peventaccurate evaluation of edema      
Interpretation of results:
Category 2 (irritant)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item produced a primary irritation index of 4.0 and was classified as a moderate irritant to rabbit skin according to the Draize classification, when applied under a semi-occlusive dressing for 4 hours. 3 minute and 1 hour exposure of the test item to the intact skin of one rabbit produced no corrosive effects.

According to CLP criteria the the scores for erythema and/or edema are below the score of 2.3 in at least 2 of 3 animals after 4 h of exposure. The effects were reversible within the 14 day observation period. However as skin discoloration and scab formation occurred during the observation period, a classification as category 2 irritant is proposed.

According to Dir. 67/548/EC and adaptations the scores for erythema and/or edema were 2 or greater in 2 of three animals and the substance is consequently classified as irritant to skin R38.
Executive summary:

A rabbit dermal irritation study conducted by the method of OECD 404 was conducted using New Zealand White Rabbits. A single animal showed no corrosive effects from a 3 -minute or a 1 hour semi-occluded application of test article to intact skin. Scores for erythema increased from 24 to 72 hours, with maximum scores of 2 at the 72 hour reading. Blanching of the skin was noted at 72 hours in the 3 minute and 72 hour sites. Crusting was seen at 7 days after the 1 hour exposure, but other than glossy skin at the 3 minute exposure site, the skin had returned to normal by 14 days. Oedema readings increased to a maximum of 2 at 72 hours in the 3 minute and 1 hour exposure sites. Edema readings could not be assessed at 7 days in the 1 hour exposure site. Readings for edema were normal at 14 days in both exposure sites.

In the 4 hour exposure portion of the experiment, an additional 2 rabbits were added to the original for a total of 3 rabbits. A single 4 hour exposure of the test item to intact skin on these 3 animals produced well-defined erythema and slight to moderate edema. Maximum scores for erythema of 2 were seen for all rabbits at the 24 to 72 hour readings. Other skin readings observed were brown discoloration of the epidermis, hardened dark brown colored scab, loss of skin elasticity and flexibility, undulating scab, modreate desquamation and crust formation than hindered accurate evaluation of edema and erythema in one rabbit at 7 days. Two rabbits had normal readings except for some desquamation at 7 days, and the thrid rabbit had normal readings at 14 days. In t he opinion of the Study Director, these observations did not indicate corrosion.

Based on a Primary Irritation Index of 4.0, the test article was classified as a moderate irritant to rabbit skin according to the Draize classification scheme.

According to CLP (Reg. 1907/2006 and amendments) criteria the the scores for erythema and/or edema are below the score of 2.3 in at least 2 of 3 animals after 4 h of exposure. The effects were reversible within the 14 day observation period. However as skin discoloration and scab formation occurred during the observation period, as classification as category 2 irritant is proposed.

According to Dir. 67/548/EC and adaptations the scores for erythema and/or edema were 2 or greater in 2 of three animals and the substance is consequently classified as irritant to skin R38.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
26 August 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Followed established Guideline and GLP requirements
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
In vitro test in step wise procedure of Guideline 405
Qualifier:
according to
Guideline:
other: OECD No. 437 (2009) "Bovine Corneal Opacity and Permeability Assay"
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
other: bovine corneas
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: local abbatoir
- Age at study initiation: adult cattle, eyes obtained from freshly slaughtered animals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): eyes transported in Hanks' Balanced Salt Solution supplemented by Penicillin/Streptomycin and placed on ice packs. Eyes refrigerated on arrival to lab, and used within 24 hours.


Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL test item or control item applied to each cornea.
- Concentration (if solution): Negative control was 0.9% sodium chloride solution.

Duration of treatment / exposure:
After test item or control item applied to cornea in the BCOP holder and the holder tilted to assure uniform applicaiton to the cornea, the holder containing the cornea was incubated at 32 +/- 1 degrees C for 10 minutes. The cornea was then rinsed three times with complete Minimum esssential medium (MEM) containing phenol red before a final rinse with complete MEM.
Observation period (in vivo):
A pre-treatment opacity reading of each cornea was taken before the exposure period. A post-treatment opacity reading was taken and each cornea observed visually after the rinsing with complete MEM. The holders were then incubated at 32 +/- 1 degrees C for 120 minutes +/- 10 minutes. After that incubation, a final opacity reading was taken and each cornea visually observed.Following the final opacity reading, the medium was removed from the anterior chamber and replaced with 1 ml sodium fluorescein (4 mg/ml). The holders were incubated for 90 minutes at 32 +/- 1 degrees C. Medium from the posterior chamber was used for optical density reading (a sample of 360 ul represented each cornea.
Number of animals or in vitro replicates:
Three corneas each for test item, negative control and positive control
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, after 10 minutes, the corneas were rinsed with MEM plus phenol red three times before a final rinse with complete MEM
- Time after start of exposure: 10 minutes of exposure

SCORING SYSTEM: Opacity reading using calibrated opacitometer; permeability measured by optical density at 492 nm;

TOOL USED TO ASSESS SCORE: Calibrated opacitometer; 96 well plate for measuring optical density at 429 nm.
Irritation parameter:
other: In Vitro Irritation Score (IVIS)
Basis:
other: mean value for treated cornea group
Score:
ca. 66
Remarks on result:
other: The IVIS score was greater then 55, thus the test item is a severe irritant or corrosive
Irritant / corrosive response data:
See tables in results section for individual corneal and mean treatment values for opacity, permeability, corrected values of each, and the resulting IVIS scores.

Individual and Mean Corneal Opacity Measurements

Opacity

Treatment

Cornea Number

Pretreatment/

Postreatment/Post Incubation

Post incubation - Pretreatment

Corrected Value

Negative Control

1

1

1

2

1

2

1

1

1

0

3

2

2

3

1

Mean

0.7

Positive Control

4

2

28

28

26

25.3

5

1

27

27

26

25.3

6

2

25

25

23

22.3

Mean

24.3

Test Item

7

3

5

9

6

5.3

8

1

10

13

12

11.3

9

1

4

18

17

16.3

Mean

11.0

Individual and Mean Permeability Measurements and Resulting IVIS

Permeability (Optical Density, OD)

Treatment

Cornea Number

Corrected Value

In vitro Irritancy Score (IVIS)

Negative Control

1

0.057

2

0.127

3

0.056

Mean

0.080

1.9

Positive Control

4

2.860

2.780

5

2.650

2.570

6

3.350

3.270

Mean

2.873

67.4

Test Item

7

4.545

4.465

8

3.665

3.585

9

3.025

2.945

Mean

3.665

66.0

Corneal Epithelium Post Treatment and Post Incubation

Observation

Treatment

Cornea Number

Post Treatment

Post Incubation

 

Negative Control

1

Clear

Clear

 

2

Clear

Clear

 

3

Clear

Clear

 

Positive Control

4

Cloudy

Cloudy

 

5

Cloudy

Cloudy

 

6

Cloudy

Cloudy

 

Test Item

7

Clear

Cloudy

 

8

Cloudy

Cloudy

 

9

Clear

Cloudy

 

Interpretation of results:
highly irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: Criteria in OECD No. 437
Conclusions:
Following assessment of all endpoint data in the Bovine Corneal Opacity and Permeability Assay, the test item was considered to have the potential to cause severe ocular irritancy in vivo. The in vivo eye irritation study is therefore not needed.

According to CLP (Reg. 1907/2006 and amendments) the substance is classified as causing irreversible eye effects (category 1) based on this in vitro result.

(Reg. 1907/2006 and amendments) the substance is classified as posing the risk of serious damage to eyes (R41).
Executive summary:

Following assessment of all endpoint data in the Bovine Corneal Opacity and Permeability Assay, the test item was considered to have the potential to cause severe ocular irritancy in vivo. The in vivo eye irritation study is therefore not needed. The In Vitro Irritancy Score, calculated on changes in opacity and permeability (optical density), was 66.0 for the test item compared to 67.4 for the positive control item (ethanol). According to the OECD Guideline No. 437, an IVIS of 55 or greater is interpreted as the test item has the potential for severe ocular irritancy in vivo.

According to CLP (Reg. 1907/2006 and amendments) the substance is classified as causing irreversible eye effects (category 1) based on this in vitro result.

(Reg. 1907/2006 and amendments) the substance is classified as posing the risk of serious damage to eyes (R41).

Endpoint:
eye irritation
Remarks:
other: in vitro human cornea model
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2011-04-12 to 2011-04-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Follwos accepted scientific principles and conducted under GLP
Reference:
Composition 1
Qualifier:
according to
Guideline:
other: Van Goethem F, et al,"Prevalidation of a new in vitro reconstituted human cornea model to assess the eye irritating potential of chemicals," Toxicology in vitro, 20 (2006)
Principles of method if other than guideline:
The SkinEthic HCE model consists of transformed human corenal epithelial cells of the cel line HCE that form a corneal epithelial tissue (mucosa) devoid of stratum corneum, resembling, histologically, the mucosa of the human eye. The test article is applied to the culture surface at the air interface so that undiluted or end use dilutions can be teste directly. the model consists of an airlifted, living corneal tissue construct, produced in polycarbonate inserts in serm-free and chemically defined medium. The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the HCE model and are sufficiently cytotoxic to cause cell death in underlying cell layers, Cytotoxicity was determined by reduction of MTT to formazan by living cells in the test item treated tissues (quantitative measurement of cell viability) relative to the negative control). Measurement of the optical density (OD540) quantified the color change of the MTT to formazan. One tissue per treatment group was retained for possible histopatholoty.
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
other: transformed human corneal epithelial cells
Strain:
other: Cell line HCE (LSU Eye Center, New Orleans, USA
Details on test animals or tissues and environmental conditions:
TEST System: SKINETHIC HCE Model
- Source: SkinEthic Laboratories, Nice, France

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37 degrees C, 5% CO2 in air storage conditions for receipt and overnight incubation of Day 6 cultures


Study DATES: From: 12 April 2011 To: 14 April 2011
Vehicle:
unchanged (no vehicle)
Controls:
other: negative and positive control groups
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): MTT direct reduction assessment: 30 ul test item to 1 ml of 0.5 mg/mL MTT solution
Main assay: 30 ul test item or control item applied to triplicate tissues for 10 minutes
- Concentration (if solution): test item as supplied
SDS positive control prepared as 2% w/v in sterile water
Solution A negative control used as supplied
Control substances:
Negative Solution A as supplied by Test kit
Positive Sodium dodecyl sulphate (SDS) 2%w/v prepared in distilled water

Duration of treatment / exposure:
Ten minutes exposure of tissues
Observation period (in vivo):
Main test: following exposure, and tissue rinsing, each tissue was exposed to the MTT solution and incubated for 3 hours. At the end of the incubatin period, the tissues were examined, and the degree of MTT staining assessed (qualitative evaluation). Tissues were then mived to the MTT extraction plates, isopropanol applied, and the plates covered with foil and allowed to stand at room temperature overnight to extract the formazan crystals. At the end of the extractin period, extraction fluids were withdrawn and optical density (quantitative assessment of viability) made.
Number of animals or in vitro replicates:
Three tissues for treatment group - 1 tissue retained for possible histopathology
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, with Dulbecco's Phosphate Buffered Saline with Ca++ and Mg++,
- Time after start of exposure: to minutes

SCORING SYSTEM: OD540 readings used to calculate relative mean tissue viability of treated group
Relative mean tissue viability (%) = (mean OD540 of test item/mean OD540 of negative control) x 100

TOOL USED TO ASSESS SCORE: Anthos 2001 microplate reader

Classification:
Relative mean tissue viability Prediction
Tissue viability < 60% Irritant (I)
Tisue viability =/< 60% Non-irritant

Criteria for a Valid Test:
Positive control relative mean tissue viability < 60% relative to negative control treated tissues
MTT direct test shows chemical does not directly reduce MTT
Irritation parameter:
other: relative mean tissue viability (%)
Basis:
other: mean viability of treated compared to control tissues
Time point:
other: 10 minute exposure plus time for MTT expsoure, formazan extraction and OD540 readings
Score:
ca. 52.6
Irritant / corrosive response data:
See Any Other Information Section

Mean OD540 Values and Percentage Viabilities for the Negative Control, Positive Control and Test Items

Item

OD540of tissues

Mean OD540(triplicate tissues)

Relative mean viability (%)

Negative Control Item

0.895

0.879

0.887

100*

Positive Control Item

0.222

0.171

0.197

22.2

Test Item

0.517

0.417

0.467

52.6

*  mean viability of the negative control tissues is set at 100%

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: other:
Conclusions:
The test article was considered to be irritating to the eye based on the relative cell viability of < 60% compared to control tissues in the SKINETHIC Human corneal Epithelial Model assay with 10 minute exposure.
Executive summary:

The purpose of the test was to evaluate eye irritation potential of the test article using the SKINETHIC reconstructed human corneal model (HCE, SkinEthic Laboratories, Nice, France) after a treatment period of 10 minutes. The principle of the assay was based on measurement of cytotoxicity in reconstructed human corneal cell cultures following topical exposure to test item by means of colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethythiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to negative controls.   

The relative mean viability of the test article treated tissues was 52.6% after the 10 minute exposure. Quality criteria for the acceptance of the results were satisfied. The test article was considered to be irritant.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

The skin and eye irritation properties of the test material were investigated using a tiered testing strategy. From physical chemical and structural considerations an irritating or corrosive effect could not be excluded, but also not positively concluded. Therefore in vitro studies for skin and eye irritation/corrosion were performed.

The test item was assessed for skin corrosivity potential using the Corrositex assay. The method designed was compatible with OECD Guidelines for the Testing of Chemicals, No. 435 “In vitro Membrane Barrier Method for Skin Corrosion.” The assay consisted of a qualification screen to determine if the chemical could be detected by the Chemical Detection System, a categorization screen to determine test items with high or low acid/alkaline reserve and the definitive test. The test article did qualify for the detection system and was determined to be a Category 1 chemical (high acid/alkaline reserve). No breakthrough of the membrane was noted after 4 hours. It was concluded that the test article was non-corrosive under the conditions of the test.

Therefore the skin irritation potential of the test article was examined in a second in vitro test using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes and a post-exposure incubation period of 42 hours. The principle of the assay was based on measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to test item by means of the colorimetric MTT reduction assay. The relative mean viability of the test article treated tissues was 80.7% after the 15 minute exposure period. Quality criteria for the acceptance of the results were satisfied. The test article was considered to be non-irritant.

Following the negative in vitro result, a skin irritation study in rabbits was performed with the substance according to OECD 404 using New Zealand White Rabbits. A single animal showed no corrosive effects from a 3 -minute or a 1 hour semi-occluded application of test article to intact skin. Scores for erythema increased from 24 to 72 hours, with maximum scores of 2 at the 72 hour reading. Blanching of the skin was noted at 72 hours in the 3 minute and 72 hour sites. Crusting was seen at 7 days after the 1 hour exposure, but other than glossy skin at the 3 minute exposure site, the skin had returned to normal by 14 days. Oedema readings increased to a maximum of 2 at 72 hours in the 3 minute and 1 hour exposure sites. Edema readings could not be assessed at 7 days in the 1 hour exposure site. Readings for edema were normal at 14 days in both exposure sites. In the 4 hour exposure portion of the experiment, an additional 2 rabbits were added to the original for a total of 3 rabbits. A single 4 hour exposure of the test item to intact skin on these 3 animals produced well-defined erythema and slight to moderate edema. Maximum scores for erythema of 2 were seen for all rabbits at the 24 to 72 hour readings. Other skin readings observed were brown discoloration of the epidermis, hardened dark brown colored scab, loss of skin elasticity and flexibility, undulating scab, modreate desquamation and crust formation that hindered accurate evaluation of edema and erythema in one rabbit at 7 days. Two rabbits had normal readings except for some desquamation at 7 days, and the thrid rabbit had normal readings at 14 days. In the opinion of the Study Director, these observations did not indicate corrosion. Although the results indicate a borderline case with regard to classification as irritant it is concluded that the substance should be considered as irritating to skin and classified accordingly.

The eye irritation potential was concluded based on the results of 2 in vitro experiments.

The first experiment was based on the SKINETHIC reconstructed human corneal model (HCE, SkinEthic Laboratories, Nice, France) after a treatment period of 10 minutes. The principle of the assay was based on measurement of cytotoxicity in reconstructed human corneal cell cultures following topical exposure to test item by means of colorimetric MTT reduction assay.  

The relative mean viability of the test article treated tissues was 52.6% after the 10 minute exposure. Based on this result the test article was considered to be irritant to eyes.

The second experiment was the Bovine Corneal Opacity and Permeability Assay. The In Vitro Irritancy Score, calculated on changes in opacity and permeability (optical density), was 66.0 for the test item compared to 67.4 for the positive control item (ethanol). According to the OECD Guideline No. 437, an IVIS of 55 or greater is interpreted as a potential for severe ocular irritancy in vivo. Based on this study it is concluded that the substance may cause irreversible eye effects.


Effects on skin irritation/corrosion: irritating

Effects on eye irritation: highly irritating

Justification for classification or non-classification

Skin irritation: The substance is classified based on the results of the in vivo skin irritation study in rabbits.

According to CLP (Reg. 1907/2006 and amendments) criteria the the scores for erythema and/or edema are below the score of 2.3 in at least 2 of 3 animals after 4 h of exposure. The effects were reversible within the 14 day observation period. However as skin discoloration and scab formation occurred during the observation period, a classification as category 2 irritant is proposed.

According to Dir. 67/548/EC and adaptations the scores for erythema and/or edema were 2 or greater in 2 of three animals and the substance is consequently classified as irritant to skin R38.

Eye irritation: Based on the result of an in vitro Bovine Corneal Opacity and Permeability Assay, the substance is classified as causing irreversible eye effects (category 1) according to CLP (Reg. 1907/2006 and amendments).

According to Dir. 67/548/EC and adaptations

the substance is classified as posing the risk of serious damage to eyes (R41).