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Description of key information

Skin irritation: in vivo: The test material should be considered to be a category 1C skin corrosive, due to necrosis seen in more than 1 animal.

Skin corrosion: in vitro / ex vivo: The test item was considered to be non-corrosive to the skin. However, due to the presence of in vivo data, the in vivo study takes precedence over the in vitro result.

Skin irritation: in vitro/ ex vivo: An in vitro skin irritation study does not need to be conducted if an in vivo study is available.

An in vivo eye irritation study for this test item is available, however due to the inconsistency of effects between animals and the scoring, with some animals showing slight redness and chemosis and some animals showing necrosis and ulceration which appears to reverse by day 7, this study is not considered reliable for classification. However this study does indicate that the test item may cause eye damage/irritation.

In accordance with Regulation (EC) No 1907/2006 section 8.2 column 2, a serious eye damage/irritation study does not need to be conducted if the substance is classified as skin corrosion, leading to classification as serious eye damage (category 1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Experimental start date 25 October 2016 Experimental completion date 04 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
OECD Guideline for the Testing of Chemicals No. 431 In Vitro Skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method (28 July 2015).
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Method B.40bis
Version / remarks:
Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Physical state/Appearance: Amber coloured gelatinous solid
Batch: E00268-014-212
Purity: 100%
Expiry Date: 10 June 2018
Storage Conditions: Room temperature in the dark
Test system:
human skin model
Source species:
human
Cell type:
other: The target cells are epithelial
Cell source:
other: EpiDerm™ Human Skin Model
Source strain:
other: EpiDerm™ Human Skin Model
Details on animal used as source of test system:
EpiDerm™ Human Skin Model
Justification for test system used:
The purpose of this test was to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.
Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item-treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier : MatTek
Date received: 01 November 2016
EpiDermTM Tissues (0.63cm2) lot number: 23372
Assay Medium lot number : 1027162SA
Upon receipt of the EpidermTM tissues, the sealed 24 well plate was stored in a refrigerator until use.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
The test item was pressed flat and approximately 8 mm diameter discs were cut out. The discs had an average weight of approximately 70 mg.
Duration of treatment / exposure:
exposure periods of 3 and 60 minutes
Number of replicates:
6 well plates
Controls:
yes, concurrent positive control
yes, concurrent negative control
Irritation / corrosion parameter:
other: Mean OD562 values
Run / experiment:
3 minute
Value:
92.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
The mean OD562 for the negative control treated tissues was 1.819 for the 3 Minute exposure period and 1.877 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied. The relative mean tissue viability for the positive control treated tissues was 3.8 % relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied. In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.
Irritation / corrosion parameter:
other:
Run / experiment:
60 minute
Value:
94.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
The mean OD562 for the negative control treated tissues was 1.819 for the 3 Minute exposure period and 1.877 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied. The relative mean tissue viability for the positive control treated tissues was 3.8 % relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied. In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Direct MTT Reduction

The MTT solution containing the test item did not turn blue/purple.  This was taken to indicate the test item did not reduce MTT.

Assessment of Colour Interference with the MTT endpoint

The solution containing the test item was a yellow colour.  This colour was attributed to the intrinsic colour of the test item itself.  It was therefore considered unnecessary to run colour correction tissues.

The relative mean viabilities for each treatment group were as follows:

 Exposure Period        Percentage Viability
   Negative Control  Positive Control

 Test Item

 3 Min  100*  4.9  92.7
 60 Min 100*   3.8  94.1

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

Quality Criteria

The mean OD562 for the negative control treated tissues was 1.819 for the 3 Minute exposure period and 1.877 for the 60 Minute exposure period.  The negative control acceptance criteria were therefore satisfied.

The relative mean tissue viability for the positive control treated tissues was 3.8 % relative to the negative control following the 60 Minute exposure period.  The positive control acceptance criterion was therefore satisfied.

In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%.  The acceptance criterion was therefore satisfied.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be non-corrosive to the skin.
Executive summary:

Introduction

The purpose of this test was to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item-treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Methods

Duplicate tissues were treated with discs of the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT loading. After MTT-loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 ¿L samples were transferred to the appropriate wells of a pre-labeled 96 well plate. The optical density (OD) was measured at 562 nm (OD562).

Data are presented in the form of percentage viability (MTT reduction in the test item-treated tissues relative to negative control tissues).

Results

The relative mean viabilities for each treatment group were as follows:

Exposure Period                                             Percentage Viability

                                              Negative Control             Positive Control               Test Item

3 minute                                            100*                                    4.9                        92.7

60 minute                                         100*                                    3.8                        94.1

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

Quality Criteria

The quality criteria required for acceptance of results in the test were satisfied.

The test item was considered to be non-corrosive to the skin.

Conclusion

The test item was considered to be non-corrosive to the skin.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 27, 1987 to February 26, 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
other: Not reported
Deviations:
not applicable
Principles of method if other than guideline:
The primary skin irritation potential of the test material was evaluated following its application to the clipped backs of 6 New Zealand White rabbits. The test material was administered as a single 0.5 g dose introduced under a gauze patch, moistened with saline and secured with tape. The patch was loosely held in contact with the skin by means of an occlusive dressing. The dressing remained in place for 4 hours. After exposure was completed, the dressing and gauze were removed and the test site was graded for erthema, edema and other signs of dermal irritation at 45 m, 24, 48 and 72 h following patch removal. Dermal readings were also made on Day 7. All dermal scoring was made according to the Draize Method of scoring.
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch No. 1

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

OTHER SPECIFICS: The test material was a waxy off-white solid which was assumed to be 100 % pure for the purpose of dosing.
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Hazelton Reserach Products, Inc., Denver, Pennsylvania
- Age at study initiation: Approximately 13 weeks
- Weight at study initiation: 2.29 to 3.15 kg
- Housing: Individually in suspended stainless steel caging
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 22 d

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 65 to 70
- Humidity (%): 40 to 60
- Photoperiod (hrs dark / hrs light): 12/12 by automatic timer

IN-LIFE DATES: From: October 27, 1987 To: November 3, 1987
Type of coverage:
occlusive
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:
7 d
Number of animals:
3 males, 3 females
Details on study design:
- Preparation of animals: The day prior to topical administration of the test material, the hair of each rabbit on the dorsal surface from the shoulder region to the lumbar region was closely clipped with an electric clipper. The skin was left intact. Elizabethan-type collares were placed around the neck of each rabbit at this time to acclimate them to wearing collars. Animals were reclipped as needed for dermal evaluations.
- Administration of test material: The test material was administered as a single 0.5 g dose, moistened with 0.5 mL saline and introduced under a gauze patch which was loosely held in contact with the skin by means of an occlusive dressing for the duration of the exposure period. After approximately 4 h exposure, the dressing and gauze patch were removed. Residual test material was removed using distilled water and paper towels. Collars were removed after the exposure period.
- Experimental evaluation: The animals were examined for viability twice daily on weekdays and once daily on weekends. Dermal responses were evaluated approximately 45 minutes, 24, 48 and 72 h following patch removal, and on Day 7. All scoring was made according to the Draize Method of Scoring. Body weights were recorded on the day of dosing (Day 0) and on Day 7. After the Day 7 observations and terminal weighings, all rabbits were sacrified and discarded without further examination.
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
other: All animals
Score:
4.5
Remarks on result:
probability of moderate irritation
Irritation parameter:
erythema score
Basis:
other: mean score for animal 1, 2 and 6
Time point:
24/48/72 h
Score:
2.66
Irritation parameter:
erythema score
Basis:
other: mean score for animal 3
Time point:
24/48/72 h
Score:
2
Irritation parameter:
erythema score
Basis:
other: mean score for animal 4
Time point:
24/48/72 h
Score:
2.33
Irritation parameter:
erythema score
Basis:
other: mean score for animal 5
Time point:
24/48/72 h
Score:
3
Irritation parameter:
edema score
Basis:
other: mean score for animal 1, 2 and 6
Time point:
24/48/72 h
Score:
1.66
Irritation parameter:
edema score
Basis:
other: mean score for animal 3 and 4
Time point:
24/48/72 h
Score:
1
Irritation parameter:
edema score
Basis:
other: mean score for animal 6
Time point:
24/48/72 h
Score:
2
Irritant / corrosive response data:
Topical application of the test item elicited erythema in all animals. Erythema was noted as very slight in all animals at the 45 Minute observations and increased as the study progressed. Severe erythema was noted in 5 animals at the 72 Hour observation while the remaining animal was noted with well-define erythema. Severe erythema was observed in all animals at the Day 7 observations.

Edema scores at the 45 Minute observations were limited to 2 animals who exhibited very slight edema. Edema scores increased during the test period and by termination were substantially increased. Edema was noted as very slight in 1 animal, slight in 4 animals and moderate in the remaining animal.

Supplemental dermal observations were noted in all animals during the study and included eschar, necrosis, desquamation, atonia and leathery skin. Supplemental dermal observations were limited to the 72 Hour and Day 7 observations.

Please see attached:

Table 1: Individual dermal irritation scores for all animals

Table 2: Supplemental dermal observations

Table 3: Individual animal body weights by weighing period

Appendix A: The Draize Method of Scoring used for evaluation of dermal irritation.

Interpretation of results:
Category 1C (corrosive) based on GHS criteria
Remarks:
We have concluded that there is enough evidence to consider the test substance to be classified as a skin corrosive, category 1C.
Conclusions:
The primary skin irritation potential of the test material was evaluated following its application to the clipped backs of 6 New Zealand White rabbits. The test material was administered as a single 0.5 g dose introduced under a gauze patch, moistened with saline and secured with tape. The patch was loosely held in contact with the skin by means of an occlusive dressing. The dressing remained in place for 4 hours. After exposure was completed, the dressing and gauze were removed and the test site was graded for erthema, edema and other signs of dermal irritation at 45 m, 24, 48 and 72 h following patch removal. Dermal readings were also made on Day 7. All dermal scoring was made according to the Draize Method of scoring. Topical application of the test item elicited erythema in all animals. Erythema was noted as very slight in all animals at the 45 Minute observations and increased as the study progressed. Severe erythema was noted in 5 animals at the 72 Hour observation while the remaining animal was noted with well-define erythema. Severe erythema was observed in all animals at the Day 7 observations. Edema scores at the 45 Minute observations were limited to 2 animals who exhibited very slight edema. Edema scores increased during the test period and by termination were substantially increased. Edema was noted as very slight in 1 animal, slight in 4 animals and moderate in the remaining animal. Supplemental dermal observations were noted in all animals during the study and included eschar, necrosis, desquamation, atonia and leathery skin. Due to visible nerosis was seen in at least one animal, the substance is classified as a Category 1C skin corrosive.
Executive summary:

Due to necrosis being seen in at least 1 animal, we have concluded that there is enough evidence to consider the test substance to be classified as a skin corrosive, category 1C. 

Endpoint:
skin irritation: in vitro / ex vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation: in vivo

The primary skin irritation potential of the test material was evaluated following its application to the clipped backs of 6 New Zealand White rabbits. The test material was administered as a single 0.5 g dose introduced under a gauze patch, moistened with saline and secured with tape. The patch was loosely held in contact with the skin by means of an occlusive dressing. The dressing remained in place for 4 hours. After exposure was completed, the dressing and gauze were removed and the test site was graded for erthema, edema and other signs of dermal irritation at 45 m, 24, 48 and 72 h following patch removal. Dermal readings were also made on Day 7. All dermal scoring was made according to the Draize Method of scoring. Topical application of the test item elicited erythema in all animals. Erythema was noted as very slight in all animals at the 45 Minute observations and increased as the study progressed. Severe erythema was noted in 5 animals at the 72 Hour observation while the remaining animal was noted with well-define erythema. Severe erythema was observed in all animals at the Day 7 observations. Edema scores at the 45 Minute observations were limited to 2 animals who exhibited very slight edema. Edema scores increased during the test period and by termination were substantially increased. Edema was noted as very slight in 1 animal, slight in 4 animals and moderate in the remaining animal. Supplemental dermal observations were noted in all animals during the study and included eschar, necrosis, desquamation, atonia and leathery skin. Based necrotic findings in at least 1 animal, the test material should be considered to be a category 1C skin corrosive. 

 

Skin corrosion: in vitro / ex vivo

The purpose of this test was to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item-treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

The test item was considered to be non-corrosive to the skin in vitro. However, due to the presence of in vivo data, the in vivo study takes precedence over the in vitro study, therefore the test item is classified as a skin corrosive, category 1C.

Justification for classification or non-classification

Skin irritation: in vivo: The test material should be considered to be a category 1C skin corrosive, due to necrosis seen in more than 1 animal.

CLP guidance says “A skin corrosive is considered to also cause serious eye damage which is indicated in the hazard statement for skin corrosion, H314: Causes severe skin burns and eye damage.

Thus, in this case a substance has to be classified for both classifications (Skin Corr. 1 and Eye Dam. 1) but the hazard statement H318 ‘Causes serious eye damage’ is not indicated on the label because of redundancy (CLP Article 27).”