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Description of key information

Skin irritation/corrosion: 
Skin corrosion study (in vitro, occlusive, 3 and 60 minutes), human (EPIDERM™ Human Skin Model) - GLP, OECD Guideline 431: non-corrosive to skin (relative mean tissue viability 3 min: 94.9% and 60 min: 99.6%)
Acute (single dose) eye irritation study (in vivo), rabbit (New Zealand White) - GLP, OECD Guideline 405, EU Method B5: eye irritant (category 2, H319) (overall irritation score: 12.5)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin corrosion: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28.10.2014 - 30.10. 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed by following the recommended method (OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Human Skin Model Test”) and GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
Adopted 13.4.2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP certificate is as an appendix to the study report.
Specific details on test material used for the study:
The substance is alkaline, and when in water the pH is about 11.
Test system:
human skin model
Source species:
human
Cell type:
other: The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium.
Justification for test system used:
The test model incorporates several features, which make it advantageous in the study of potential dermal corrosivity potential. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at air interface, so that undiluted and/or end use dilutions can be tested directly. 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 corrosivity potential of the test item.
Vehicle:
unchanged (no vehicle)
Details on test system:
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 Celsius degrees
- Temperature of post-treatment incubation (if applicable): After rinsing of the test item, the test plates were incubated (37 C, 5% CO2) for 3 hours with MTT. For MTT extraction, 2 mL of Isopropanol was used to completely immerse each insert. The plates were placed into a refrigerator overnight, to allow extraction to proceed.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test
item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed.
- Observable damage in the tissue due to washing: no

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Absorbency of each sample well was measured using the Anthos 2001 microplate reader
- Wavelength: 562nm (OD562)

DECISION CRITERIA
- The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. Classification of corrosivity potential was based on relative viabilities for each exposure time
according to the following prediction model:

Mean tissue viability (% negative control) Prediction: Corrosive/Non-Corrosive
3 minute exposure : <50 Corrosive

3 minute exposure : >50
60 minute exposure : <15 Corrosive

3 minute exposure : >50
60 minute exposure : >15 Non-corrosive
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of sterile distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of Potassium Hydroxide
- Concentration (if solution): 8.0 N
Duration of treatment / exposure:
3-Minute exposure period
60-Minute exposure period
Duration of post-treatment incubation (if applicable):
After rinsing of the test item, the test plates were incubated (37 C, 5% CO2) for 3 hours with MTT. For MTT extraction, 2 mL of Isopropanol was used to completely immerse each insert. The plates were placed into a refrigerator overnight, to allow extraction to proceed.
Number of replicates:
2x 3-Minute exposure
2x 60-Minute exposure
Species:
human
Details on test animals or test system and environmental conditions:
TEST TISSUE:
Tissue: EPIDERM™ Reconstructed Human Epidermis Model Kit
Supplier: MatTek Corporation, Ashland, MA, USA
Date received: 28 October 2014
EpiDermTM Tissues (0.5cm2) lot number : 19690, Kit D
Assay Medium lot number : 102314TMD

Upon receipt of the EpidermTM tissues, the sealed 24-well plate was placed into a refrigerator overnight.
Type of coverage:
occlusive
Preparation of test site:
other: Not applicable. Skin tissue.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of sterile distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of Potassium Hydroxide
- Concentration (if solution): 8.0 N
Duration of treatment / exposure:
Two exposure periods:
- 3 minutes
- 60 minutes
Observation period:
Three hours incubation with MTT (37 C, 5% CO2), after rinsing the tissues from the test material with the help of Dulbecco’s phosphate buffered saline (DPBS). For MTT extraction, 2 mL of Isopropanol was used to completely immerse each insert. The plates were placed into a refrigerator overnight, to allow extraction to proceed.
Number of animals:
Two tissue samples per exposure period.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper.
- Time after start of exposure: Three minutes or 60 minutes.

SCORING SYSTEM:
Quantitative MTT Assessment (percentage tissue viability)
The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water.

The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD562 of test item / mean OD562 of negative control) x 100

Classification of corrosivity potential was based on relative viabilities for each exposure time
according to the following prediction model:

Mean tissue viability (% negative control) Prediction (Corrosive/Non-Corrosive)
- 3 minute exposure : <50 Corrosive
-3 minute exposure : >50 and 60 minute exposure : <15 Corrosive
- 3 minute exposure : >50 and 60 minute exposure : >15 Non-corrosive

Quality Criteria
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Negative Control:
The assay establishes the acceptance criterion for an acceptable test if the mean optical density for the negative control treated tissues is 0.8 or higher.
Positive Control:
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤ 30% relative to the negative control treated tissues following the 3-Minute exposure period.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-Minute exposure
Value:
94.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60-Minute exposure
Value:
99.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue. This was taken to indicate the test item did not reduce MTT.
- Colour interference: No. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean OD562 for the negative control treated tissues was 1.826 for the 3-Minute exposure period and 2.243 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.

- Acceptance criteria met for positive control: Yes. The relative mean tissue viability for the positive control treated tissues was 8.5 % relative to the negative control treated tissues following the 3-Minute exposure period. The positive control acceptance criterion was therefore satisfied.

Table 1. Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item Exposure Time  Mean OD562** % Viability
Negative Control  3 minute 1.826 100*
60 minute  2.243 100*
Positive Control  3 minute 0.156 8.5
60 minute  0.088 3.9
Test Item  3 minute 1.732 94.9
60 minute  2.235 99.6

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

** = Mean of EpiDerm tissues tested in duplicate

Interpretation of results:
GHS criteria not met
Remarks:
According to GHS, the substance is not classified as corrosive to the skin.
Conclusions:
The test item was considered to be non-corrosive to the skin. Therefore, it is not classified as corrosive to skin.
Executive summary:

Introduction

The purpose of this test is 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 the test item for exposure periods of 3 and 60 minutes. 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 of the test item treated tissues were: 60 minutes exposure : 99.6 % 3 minutes exposure : 94.9% Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
5.5.2015-9.6.2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance with recommended Guidelines (OECD Guideline for the Testing of Chemicals No. 405 “Acute Eye Irritation/Corrosion” (adopted 02 October 2012) and Method B5 Acute Toxicity (Eye Irritation) of Commission Regulation (EC) No. 440/2008) and GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
Adopted 02 October 2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
(EC) No. 440/2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Certificate as an attachment to the study report
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues 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: 2.74 or 3.29 kg
- Housing: The animals were individually housed in suspended cages.
- Diet (e.g. ad libitum): 2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 5 days

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

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL (weight approximately 95 mg, as measured by gently compacting the required volume into an adapted syringe)


Duration of treatment / exposure:
1 treatment. The test item was left in the eyefor the 14-day assessment period.
Observation period (in vivo):
14 days. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. Additional observations were made on Days 7 and 14 to assess the reversibility of the ocular effects. Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.
Number of animals or in vitro replicates:
2 (males)
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): No washing

SCORING SYSTEM:
The numerical values corresponding to each animal, tissue and observation time were recorded. The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of cornea involved). For each tissue the score was calculated as follows:

Score for conjunctivae = (A + B + C) x 2
Score for iris = D x 5
Score for cornea = (E x F) x 5

Using the numerical data obtained a modified version of the system described by Kay J.H. and Calandra J.C. (1962) was used to classify the ocular irritancy potential of the test item. This was achieved by adding together the scores for the cornea, iris and conjunctivae for each time point for each rabbit. The group means of the total scores for each observation were calculated. The highest of these group means (the maximum group mean score) together with the persistence of the reactions enabled classification of the eye irritancy potential of the test item.
If evidence of irreversible ocular damage is noted, the test item will be classified as corrosive to the eye.

TOOL USED TO ASSESS SCORE:
Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope
Irritation parameter:
overall irritation score
Remarks:
Considers: cornea, iris and conjunctivae
Basis:
mean
Time point:
14 d
Score:
12.5
Max. score:
110
Reversibility:
fully reversible within: 14 days
Remarks on result:
positive indication of irritation
Remarks:
Iridial inflammation was noted in one treated eye and moderate conjunctival irritation was noted in both treated eyes. No corneal effects were noted during the study. The effects were fully reversible.
Irritation parameter:
cornea opacity score
Remarks:
Considers: degree of opacity, area of cornea involved
Basis:
animal #1
Remarks:
Male
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: no indication of irritation
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Remarks:
Considers: degree of opacity, area of cornea involved
Basis:
animal #2
Remarks:
Male
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: no indication of irritation
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Remarks:
Male
Time point:
24/48 h
Score:
5
Max. score:
5
Reversibility:
fully reversible within: 72 hours
Remarks on result:
positive indication of irritation
Remarks:
Iridial inflammation was noted in one treated eye.
Irritation parameter:
iris score
Basis:
animal #2
Remarks:
Male
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: no indication of irritation
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Remarks:
Considers redness, chemosis and discharge
Basis:
animal #1
Remarks:
Male
Time point:
24/48/72 h
Score:
10.6
Max. score:
12
Reversibility:
fully reversible within: 14 days
Remarks on result:
positive indication of irritation
Remarks:
Moderate conjunctival irritation was noted in the treated eye.
Irritation parameter:
conjunctivae score
Remarks:
Considers redness, chemosis and discharge
Basis:
animal #2
Remarks:
Male
Time point:
other: 1 h
Score:
12
Max. score:
12
Reversibility:
fully reversible within: 14 days
Remarks on result:
positive indication of irritation
Remarks:
Moderate conjunctival irritation was noted in the treated eye.
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
Male
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 14 days
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
Male
Time point:
other: 1 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 14 days
Irritant / corrosive response data:
No corneal effects were noted during the study.

Iridial inflammation was noted in one treated eye at the 24 and 48-Hour observations.

Moderate conjunctival irritation was noted in both treated eyes 1 and 24 hours after treatment. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye at the 48 and 72-Hour observations with minimal conjunctival irritation noted in both treated eyes at the 7-Day observation.

Both treated eyes appeared normal at the 14-Day observation. See Tables 1 and 2, Any other information on results incl. tables.
Other effects:
No other effects. Both animals showed expected gain in body weight during the study (see Table 3, Any other information on results incl. tables).

Table 1. Individual Scores and Individual Total Scores for Ocular Irritation

Number and Sex 75059 Male 75072 Male
Initial pain reaction = 0 Initial pain reaction = 0
Time After Treatment 1 hour 24 hours 48 hours 72 hours 7 days 14 days 1 hour 24 hours 48 hours 72 hours 7 days 14 days
CORNEA                        
E = Degree of Opacity 0 0 0 0 0 0 0 0 0 0 0 0
F = Area of Cornea Involved 0 0 0 0 0 0 0 0 0 0 0 0
Score (E x F) x 5 0 0 0 0 0 0 0 0 0 0 0 0
IRIS                        
D 0 1 1 0 0 0 0 0 0 0 0 0
Score (D x 5) 5
CONJUNCTIVAE                        
A = Redness 2 2 2 2 1 0 2 2 2 2 1 0
B = Chemosis 2 2 2 2 1 0 2 1 1 1 1 0
C = Discharge 2 2 1 1 0 0 2 1 0 0 0 0
Score (A + B + C) x 2 12 12 10 10 4 0 12 8 6 6 4 0
Total Score 12 17 15 10 4 0 12 8 6 6 4

0

Initial pain reaction 0 = No response: No initial pain

Cornea: 

(E) Degree of Opacity (most dense area used)

0 = No opacity

Iris:

(D) Values

0 = Normal

1 = Folds above normal, congestion, swelling, circumcorneal injection (any or all of these or combination of any thereof) iris still reacting to light (sluggish reaction is positive)

Conjunctivae:

(A) Redness (refers to palpebral and bulbar conjunctivae excluding cornea and iris)

0 =Vessels normal

1 = Vessels definitely injected above normal

2 = More diffuse, deeper crimson red, individual vessels not easily discernible

(B) Chemosis

0 = No swelling

1 = Any swelling above normal (includes nictitating membrane)

2 = Obvious swelling with partial eversion of lids

(C) Discharge

0 = No discharge

1 = Any amount different from normal (does not include small amounts observed in inner canthus of normal animals)

2 = Discharge with moistening of the lids and hairs just adjacent to lids

Table 2. Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbit Number and Sex Individual Total Scores At:
1 hour 24 hours 48 hours 72 hours 7 days 14 days
75059 Male 12 17 15 10 4 0
75072 Male 12 8 6 6 4 0
Group Total 24 25 21 16 8 0
Group Mean Score 12.0 12.5 10.5 8.0 4.0 0.0

Table 3. Individual Body Weights and Body Weight Change

Rabbit Number and Sex Individual Body Weight (kg) Body Weight Change (kg)
Day 0 Day 3
75058 Male 2.74 3.17 0.43
75063 Male 3.29 3.54 0.25
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Remarks:
Eye irrit. 2 H319. Criteria used for interpretation of results: EU
Conclusions:
The test results are sufficient to make conclusions related to the eye irritation effects of the test item.

The test item produced a maximum group mean score of 12.5 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system. According to EC No 1272/2008 the results indicate that the test item is an eye irritant (category 2, H319).
Executive summary:

Introduction

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit.

Results

A single application of the test item to the non-irrigated eye of two rabbits produced iridial inflammation and moderate conjunctival irritation. Both treated eyes appeared normal at the 14-Day observation.

Conclusion

The test item produced a maximum group mean score of 12.5 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

There are no data gaps in irritation/corrosion. Even though there is no human data available on the hazards of the substance for eye irritation, there is no reason to believe that the observed results in the acute eye irritation study in the New Zealand White strain rabbit would not be relevant to humans. Skin corrosion and skin irritation are studied on human skin and epidermis models.

No adverse effects severe enough to cause classification of the test item were observed in either the skin corrosion study or the skin irriation study. However, in the acute eye irritation study, the test item produced a maximum group mean score of 12.5 and was classified as an eye irritant (category 2, H319) according to the CLP Regulation (EC No 1272/2008).



Justification for selection of skin irritation / corrosion endpoint:
The in vitro skin corrosion study was chosen over the in vitro skin irritation study, as it featured a higher amount of the test item applied and two different exposure times investigated, and gave more varied results in terms of relative mean viabilities of the test item treated tissues. In both studies no adverse effects were observed.

Justification for selection of eye irritation endpoint:
The in vivo acute eye irritation study was chosen over the in vitro eye irritation study because the results of the in vivo study are sufficient to make conclusions related to the test items eye irritation effects. The results of the in vitro study have identified the test item as not causing serious eye damage, but they do not permit conclusion that the test item does not require classification for eye irritation.

Effects on eye irritation: irritating

Justification for classification or non-classification

The test item is not classified as irritant or corrosive to the skin, but is classified as an eye irritant (category 2, H319).

Skin corrosion: the test item was considered to be non-corrosive to the skin, as it did not turn the MTT solution blue and the OD562 values were well above the limits for the prediction levels. The test item was also classified as non-irritant with regards to skin irritation.

Eye irritation: the test item produced a maximum group mean score of 12.5 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system. According to the CLP Regulation (EC No 1272/2008) the results indicate that the test item is an eye irritant (category 2, H319).

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