Registration Dossier

Administrative data

Description of key information

Skin irritation: The test substance is not considered to be irritating to skin.

Eye irritation:  Using the Skin Ethic reconstructed Human Corneal Epithelium model, the relative mean viability of the test item treated tissues after a 10-minute exposure period was 2.9 %.

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:
2 August to 9 August 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards with acceptable restrictions.
Qualifier:
according to guideline
Guideline:
other: U.S. Federal Register Test (1964)
Deviations:
no
Principles of method if other than guideline:
The rabbit was securely restrained by a technician. The left side of the shaved dorsum was abraded by drawing the tip of a lancet across the skin forming a lattice pattern with parallel abrasions at intervals of 5- 10 mm. The abrasions were such as the break the stratum corneum of the epidermis without damaging the underlying tissues or causing bleeding. The right of the shaved site was left intact.
GLP compliance:
no
Remarks:
Study predates GLP.
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Morton Commercial Rabbits, Parsonage Farm, Stansted, Essex, England
- Age at study initiation: 2.5- 3 months
- Weight at study initiation: 2.2- 3.3 kg
- Housing: animals were individually housed in suspended galvanised and stainless steel cages in batteries manufactured by Cope & Cope Limited. The cages measured 0.6 x 0.6 x 0.4 m high and were fitted with mesh floors and automatic watering. A sledge running the length if the battery removed waste matter from an undertray beneath the cage floors, as required.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 °C ± 2 °C
- Humidity (%): 50 % ± 10 %
- Air changes (per hr): 17
- Photoperiod (hrs dark / hrs light): 14/ 10
Type of coverage:
occlusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
0.5 mL of test material was impregnated onto unmedicated gauze patches (25 x 25 mm) and were applied, 1 patch to each side of the abraded and intact treatment areas.
Duration of treatment / exposure:
23 h
Observation period:
24 h, 72 h and 7 d
Number of animals:
6
Details on study design:
TEST SITE
- Type of wrap if used: patches were covered by slightly larger sheets of aluminium foil, and were kept in place by an impermeable bandage of a plastic self-adhesive dressing which was wrapped twice around the trunk of the rabbit with sufficient tension to ensure the test material was in close contact to the skin.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): excess test material was removed by gently washing with warm water ans drying the site with paper towels.
- Time after start of exposure: 23 h

SCORING SYSTEM: reactions of the intact and abraded test sites were assessed according to the criteria of Draize.
Irritation parameter:
erythema score
Basis:
mean
Remarks:
Animal 1
Time point:
other: 24 h, 72 h
Score:
0
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal 2
Time point:
other: 24 h, 72 h
Score:
0.5
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal 3
Time point:
other: 24 h, 72 h
Score:
0
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal 4
Time point:
other: 24 h, 72 h
Score:
0
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal 5
Time point:
other: 24 h, 72 h
Score:
0
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal 6
Time point:
other: 24 h, 72 h
Score:
0.5
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal 1
Time point:
other: 24 h, 72 h
Score:
0
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal 2
Time point:
other: 24 h, 72 h
Score:
0.5
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal 3
Time point:
other: 24 h, 72 h
Score:
0.5
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal 4
Time point:
other: 24 h, 72 h
Score:
0
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal 5
Time point:
other: 24 h, 72 h
Score:
0
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal 6
Time point:
other: 24 h, 72 h
Score:
0.5
Irritant / corrosive response data:
Upon single dermal application of the test material, barely perceptible and/or well-defined erythematous responses were provoked on intact and/or abraded skin in 3 animals at 24 h. Four animals displayed slight oedematous responses on intact and/or abraded skin at the 24-h reading. The responses diminished in intensity so that, by 72 h, 2 animals displayed barely perceptible erythematous responses and 1 very slight oedematous response. One animal displayed discolouration of the abraded skin at 24 and 72 h. All animals displayed loss of pliability of intact and abraded skin. On Day 8 one animal displayed severe erythema with loss of flexibility and discolouration of intact and abraded skin; 1 animal displayed moderate to severe erythema to severe erythema and exfoliation on intact and abraded skin and 2 animals displayed exfoliation on intact and abraded skin.

Scoring of irritance responses elicited by single dermal application of 0.5 mL of test material under a 24 -h occlusive bandage

Animal number & sex

Type of response

Score at 24 hours

Score at 72 hours

Overall score

Score at Day 7

Intact skin

Abraded skin

Intact skin

Abraded skin

Intact skin

Abraded skin

3TA 155 ♂

Erythema

0

2+

0*

0*

1.00

4

4

Oedema

0

2

0

0

0

0

3TA 157 ♂

Erythema

0

0

1*

1*

1.00

0*

0*

Oedema

1

1

0

0

0

0

3TA 158 ♂

Erythema

0

2

0*

1*

1.50

0

0*

Oedema

0

1

1

1

0

0

3TA 159 ♂

Erythema

0

0

0*

0*

0

0ε

0ε

Oedema

0

0

0

0

0

0

3TA 160 ♂

Erythema

0

0

0*

0*

0.25

0ε

0ε

Oedema

0

1

0

0

0

0

3TA 161 ♂

Erythema

1

1

0+*

0+*

1.00

3ε

3ε

Oedema

1

1

0

0

0

0

+- Discolouration

*- Loss of pliability

ε- Exfoliation

α- Loss of flexibiility

Interpretation of results:
other: CLP criteria not met
Conclusions:
The test material was assessed for irritance responses during a single application of the test material on the intact and abraded skin of the dorsum of rabbits. The test substance is not considered to be irritating to skin according to the classification criteria of CLP Regulation (EC) no. 1272/2008
Executive summary:

The test material was assessed for irritance responses during a single application of the test material on the intact and abraded skin of the dorsum of rabbits. 0.5 mL of test material was impregnated onto unmedicated gauze patches and were applied, 1 patch to each side of the abraded and intact treatment areas. Upon single dermal application of the test material, barely perceptible and/or well-defined erythematous responses were provoked on intact and/or abraded skin in 3 animals at 24 h. Four animals displayed slight oedematous responses on intact and/or abraded skin at the 24-h reading. The responses diminished in intensity so that, by 72 h, 2 animals displayed barely perceptible erythematous responses and 1 very slight oedematous response. One animal displayed discolouration of the abraded skin at 24 and 72 h. All animals displayed loss of pliability of intact and abraded skin. On Day 8 one animal displayed severe erythema with loss of flexibility and discolouration of intact and abraded skin; 1 animal displayed moderate to severe erythema to severe erythema and exfoliation on intact and abraded skin and 2 animals displayed exfoliation on intact and abraded skin. Under the conditions of this study the test substance is not considered to be irritating to skin.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 April 2012 to 05 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study.
Qualifier:
according to guideline
Guideline:
other: SkinEthic reconstructed Human Corneal Epithelium model (HCE, SkinEthic Laboratories, Nice France) (10 minute exposure)
Principles of method if other than guideline:
The SkinEthic HCE model consists of transformed human corneal epithelial cells of the cell line HCE that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye. The test item is applied directly to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The model consists of an airlifted, living, corneal tissue construct, produced in polycarbonate inserts in serum-free and chemically defined medium.

The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic HCE model and are sufficiently cytotoxic to cause cell death in the underlying cell layers.

Cytotoxicity was determined by the reduction of MTT to formazan by viable cells in the test tiem treated tissues (quantitative measurement of tissue viability) relative to the negative control.
GLP compliance:
yes (incl. QA statement)
Details on test animals or tissues and environmental conditions:
SkinEthic HCE Model (0.5 cm2)
Supplier: SkinEthic Laboratories, Nice, France
Date received: 03 April 2012

On arrival, the SkinEthic HCE tissues (Day 6 cultures), were stored at room temperature prior to transferring into 24-well plates designated ‘arrival plates’ containing 300 µL of maintenance medium. It was important to ensure that there were no air bubbles present under the tissue inserts. The tissues were incubated overnight at 37 °C, 5 % CO2 in air.
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
Test item: 30 µL of the test item
Negative control: 30 µL of Solution A
Positive control: 30 µL of 2 % w/v SDS
Duration of treatment / exposure:
Main test: 10 min
Number of animals or in vitro replicates:
Triplicate tissues were treated with the test item.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the relevant exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++. Rinsing was achieved by fillieng and emptying each tissue insert using a constant stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated ‘holding plate’ containing 300 µL of maintenance medium (at room temperature) until all the tissues were rinsed.
- Time after start of exposure: 10 min

SCORING SYSTEM:
The mean OD 540 values of the duplicate tissues were calculated. Each of these OD540 values had already been corrected for blanks by the microplate reader.

The relative mean tissue viability (percentage of the negative control) was calculated as follows:

Relative mean tissue viability (%)= (mean OD540 of test item/ mean OD540 of negative control) x 100

The mean tissue viability for the test item was compared to the negative control and classified according to the following:

Relative mean tissue viability Prediction
(percentage of negative control)
Tissue viability <60 Irritant (I)
Tissue viability ≥60 Non-irritant (NI)
Irritation parameter:
other: relative mean viability (%)
Run / experiment:
Mean of two tissues
Value:
2.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

Assessment of direct test item reduction of MTT

The MTT solution containing the test item remained yellow which indicated that they test item did not directly reduce MTT.

Assessment of eye irritation potential

Assessment of eye irritation potential- viability of HCE tissues

Item

OD540of individual tissue

Mean OD540

Relative mean viability (%)

Negative control

0.872

0.850

100*

0.828

Positive control

0.081

0.082

9.6

0.082

Test item

0.025

0.025

2.9

0.025

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

The relative mean viability of the test item treated tissues after a 10-min exposure period was 2.9 %.

 

It was considered unnecessary to proceed with tissue histopathology.

Assay acceptance criterion

The quality criterion required for the acceptance of results in the test was satisfied.

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Remarks:
(CLP)
Conclusions:
The purpose of this study was to determine the eye irritation potential of the test item using the Skin Ethic reconstructed Human Corneal Epithelium model after a treatment period of 10 minutes. The relative mean viability of the test item treated tissues after a 10-minute exposure period was 2.9 %. According to the study plan followed the test item was considered to be a irritant.
Executive summary:

Introduction: The purpose of the study was to determine the eye irritation potential of the test item using the SkinEthic reconstructed Human Corneal Spithelim model after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.

Methods: The experimental design of the study consists of a test for direct reduction of MTT (3 -[4,5 -dimethylthizol-2 -yl]-2,5 -tetrazolium bromide) by the test item followed by the main test.

For the main test, triplicate SkinEthic tissues were treated with 30 µl of the test item for 10 minutes. Triplicate tissues treated with 30 µl of Solution A served as the negative control and triplicate tissues treated with 30 µl of 2% w/v Sodium Dodecyl Sulphate (SDS) served as the positive control.

At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues (two per group) were taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading the reduced MTT was extracted from the tissues.

After extraction the absorbancy of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured. Teh optical density was measured at 540 nm (OD540). Data are presented in the form of percentage viability (MTT conversion relative to negative controls).

The test item was classified according to the following criteria:

i) If the percentage relative mean tissue viability was ≥60% the test item was considered to be non-irritant (NI).

ii) If the percentage relative mean tissue viability was <60% the test item was considered to be an irritant (I).

Results: The relative mean viability of the test item treated tissues after a 10 -minute exposure period as 2.9%.

It was considered unnecessary to proceed with tissue histopathology.

Quality criterion: The quality criterion required for acceptance of results in the test was satisfied.

Conclusion: The test item was considered to be an irritant (I).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Skin irritation (key study): The test material was assessed for irritance responses during a single application of the test material on the intact and abraded skin of the dorsum of rabbits. 0.5 mL of test material was impregnated onto unmedicated gauze patches and were applied, 1 patch to each side of the abraded and intact treatment areas. Upon single dermal application of the test material, barely perceptible and/or well-defined erythematous responses were provoked on intact and/or abraded skin in 3 animals at 24 h. Four animals displayed slight oedematous responses on intact and/or abraded skin at the 24-h reading. The responses diminished in intensity so that, by 72 h, 2 animals displayed barely perceptible erythematous responses and 1 very slight oedematous response. One animal displayed discolouration of the abraded skin at 24 and 72 h. All animals displayed loss of pliability of intact and abraded skin. On Day 8 one animal displayed severe erythema with loss of flexibility and discolouration of intact and abraded skin; 1 animal displayed moderate to severe erythema to severe erythema and exfoliation on intact and abraded skin and 2 animals displayed exfoliation on intact and abraded skin. Under the conditions of this study the test substance is not considered to be irritating to skin.

Eye irritation (key study): An assessment of the ocular irritation potential of the test material was conducted using the SkinEthic reconstructed Human Corneal Epithelium after a treatment of 10 minutes. Triplicate SkinEthic tissues were treated with 30 µL of the test item for 10 min. At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues (2 per group) were taken for MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) loading. Following MTT loading the reduced MTT was extracted from the tissues. After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured. The optical density was measured at 540 nm (OD540). Data are presented in the form of percentage viability (MTT conversion relative to negative controls). The relative mean viability of the test item treated tissues after a 10-min exposure period was 2.9 %. Therefore under the conditions of the study the test item is considered to be an irritant.

Bovine Corneal Opactity and Permeability Assay:

The test item produced an In Vitro Irritancy Score of 31.6.

The test item was considered not to be an ocular corrosive or severe irritant.

However, according to the latest version of BCOP (OECD Guideline No. 437) the in vitro irritancy score of 31.6 would lead to a result 'no prediction of eye irritation can be made'. Classification is therefore based on the Human Corneal Epithelium model.

Justification for classification or non-classification

Skin irritation: The available study has been assigned reliability 1 and is considered as acceptable for classification.The study showed the test material did not produce a mean value of ≥2, 3- ≤4 for erythema or edema in 2 of the 3 animals from gradings at 24 and 72 h after patch removal. Therefore, the test item can be considered to be non-classified.

Eye irritation: The key study has been assigned reliability 1 and is considered as acceptable for classification.Using the Skin Ethic reconstructed Human Corneal Epithelium model, the relative mean viability of the test item treated tissues after a 10-minute exposure period was 2.9 % and therefore the test item has been classified.