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Description of key information

Skin irritation: Skin irritation potential was evaluated in the EpiDerm™ Reconstructed Human Epidermis Model (OECD 439). Following 60 minute exposure to the undiluted test item, the mean relative absorbance values indicative of cell viability were unaltered (105.9% viability; threshold for irritancy: ≤ 50%), consequently the test item was not considered irritating to the skin.

Eye irritation: The mean values for corneal opacity, iris lesions, redness of conjunctivae and oedema of conjunctivae (chemosis) were 0.0, 0.0, 1.2 and 0.8, respectively (reading time point: 24, 48, 72h). All weak signs of irritation were reversible and 7 days after application of the test article all four animals were free of any signs of eye irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 January 2017 - 03 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Commission Regulation 440/2008, 1st ATP 2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Epidermal keratinocytes
Source strain:
other: Human
Details on animal used as source of test system:
SOURCE ANIMAL
- Source: Human
- Tissue: normal epidermal keratinocytes
Justification for test system used:
Elicited via a disturbance of the desquamation process and an inflammatory response (i.e. papules, vesicles, bullae and oedema), skin irritation requires penetration of the stratum corneum and elicitation of a biological response. Skin irritation is defined in Section 3.2.1.1 of Annex I to the CLP regulation as “[…] the production of reversible damage of the skin following the application of a test substance for up to 4 hours”

According to Annex VII of the REACH Regulation, if new test data are required these must be derived from in vitro methods only. The EpiDerm™ human skin model (OECD 439) is an accepted in vitro test method to detect skin corrosion/irritation (Category 1 or 2) and/or the absence of effects (not classified under CLP).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis
- Source: MatTek Corporation (82105 Bratislava, Slovakia)
- Tissue batch number(s): 23390
- Date of initiation of testing: 06 January 2017
- Pre-warming: Prior to exposure the EpiDerm™ tissues were inspected for quality.

EPIDERM™ QUALITY CRITERIA
- Air bubbles between agarose and insert were not >30% of the total surface
- Liquid on top of the insert was removed with sterile cotton tips
- If moisture was observed on top of the inserts after the pre-incubation or in case of visible defects the respective skin models were discarded.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: DPBS, at least 15 times
- Observable damage in the tissue due to washing: None reported
- Modifications to validated SOP: None, protocol dated 29/06/2015

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Wavelength: 570 nm
- Filter: 570 nm filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Manufacturer MTT assay, 1.581 ± 0.226 OD (acceptable range: 1.0 to 3.0)
- Barrier function: Manufacturer ET-50 assay, 6.79 hrs (acceptable range: 4.77 to 8.72 hrs)
- Contamination: Manufacturer long term antibiotic and antimycotic free culture, sterile (acceptable criteria: no contamination)

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: The test chemical is considered to be irritant to skin in accordance with UN GHS and EU CLP Category 2 if the tissue viability after exposure and post-treatment incubation is ≤ 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL (corresponding to 47 μL/cm2)
- Concentration (if solution): Undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5% SLS solution in deionised water
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
Approximately 42 hours
Number of replicates:
3 per test item treatment, positive control and negative control
Irritation / corrosion parameter:
other: relative absorbance
Run / experiment:
1
Value:
99.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: relative absorbance
Run / experiment:
2
Value:
103.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: relative absorbance
Run / experiment:
3
Value:
114.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: relative absorbance
Run / experiment:
mean
Value:
105.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.
- Colour interference with MTT: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Mean OD was 1.490 (criteria: ≥ 0.8 and ≤ 2.8)
- Acceptance criteria met for positive control: Decrease in the relative absorbance as compared to the negative control was 2.2% (criteria: ≤ 20%)
- Acceptance criteria met for variability between replicate measurements: The relative standard deviations between the % variability of the test item, the positive and negative controls in the main test ≤ 9.1% (criteria: ≤ 18%)
- Range of historical values if different from the ones specified in the test guideline: 2.76 to 6.77% range of viabilities for positive control; 1.34 to 2.00 range of absorbance for negative control
Interpretation of results:
GHS criteria not met
Conclusions:
The mean relative absorbance value of the test item, corresponding to the cell viability did not decrease (105.9%; threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.
Executive summary:

Skin irritation of the test item was evaluated with the EpiDerm Reconstructed Human Epidermis Model. Cell viability of the multi-layered tissue culture of highly differentiated epidermal keratinocytes topically exposed to the test substance was evaluated using the MTT assay, which measures the conversion of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) into a blue formazan salt.

Undiluted test item was applied to the EpiDerm tissue for 60 minutes, alongside a negative and positive control. The mean relative absorbance value of the test item, corresponding to the cell viability did not decrease (105.9%; threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

The test item passed the MTT- and the Colour Interference pre-tests. Conducted according to OECD Test Guideline 439 and GLP, the study is considered to be reliable without restriction (Klimisch 1).

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:
19 June 2000 to 27 June 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The standard information requirements for REACH Annex VII substances for serious eye damage/eye irritation can be satisfied by an assessment of the available in vivo data, physicochemical properties (i.e. acid or alkali reserve) or in vitro eye irritation studies. Conducted prior to the validation and regulatory acceptance of alternative methods, a reliable (Klimisch 1) and GLP compliant in vivo Draize test was available for the test substance and considered sufficient to fulfil the information requirement.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
1987
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
other: Chbb:HM (SPF)
Remarks:
albino
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 2.7 to 2.9 kg b.w.
- Housing: Caged individually in PPO cages (floor area: 2576 sq.cm) with perforated floor
- Diet: Pelleted complete rabbit diet, ad libitum (Altromin 2123)
- Water: Domestic quality drinking water acidified with hydrochloric acid to pH 2.5, ad libitum
- Acclimation period: At least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.1 mL
Duration of treatment / exposure:
Single exposure
Observation period (in vivo):
At 1, 24, 48 and 72 hours and 7 days after treatment
Number of animals or in vitro replicates:
4
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: Eyes were rinsed with 20 mL 0.9% sodium chloride solution
- Time after start of exposure: Eyes were rinsed 24 hours after exposure

SCORING SYSTEM:
When Fluorescein was used the cornea reactions were scored both before and alter the instillation of Fluorescein.

Cornea - Opacity degree of density (Area most dense taken for reading)
0: No ulceration or opacity.
1: Scattered or diffuse area of opacity (other than slight dulling of normal luster), details of iris clearly visible.
2: Easily discernible translucent areas, details of iris slightly obscured.
3: Nacreous areas, no details of iris visible, size of pupil barely discernible.
4: Complete corneal opacity, iris not discernible.

Area of cornea involved
1: One quarter (or less) but not zero.
2: More than 1 quarter, but less than half.
3: More than half, but less than 3 quarters.
4: More than 3 quarters, up to whole area.

Iris
0: Normal.
1: Markedly deepened folds, congestion, swelling, moderate circumcorneal injection (any of these or combination of any thereof), iris still reacts to light (sluggish reaction is positive).
2: No reaction to light, haemorrhage, gross destruction (any or all of these).

Conjunctiva redness (refers to palpebral and bulbar conjunctivae excluding cornea and Iris)
0: Vessels normal.
1: Some vessels definitely injected.
2: Diffuse, crimson red, individual vessels not easily discernible.
3: Diffuse beefy red.

Conjunctiva chemosis:
0: No swelling.
1: Any swelling above normal (includes nictitating membrane).
2: Obvious swelling with partial eversion of lids.
3: Swelling with lids about half closed.
4: Swelling with lids more than half closed.

Conjunctiva 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.
3: Discharge with moistening of the lids and hairs, and considerable area around the eye.

TOOL USED TO ASSESS SCORE: fluorescein
Irritation parameter:
cornea opacity score
Basis:
animal #1
Remarks:
(1813)
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Remarks:
(1815)
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #3
Remarks:
(1816)
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #4
Remarks:
(1817)
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Remarks:
(1813)
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Remarks:
(1815)
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #3
Remarks:
(1816)
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #4
Time point:
24/48/72 h
Score:
0
Max. score:
1
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
1
Reversibility:
fully reversible
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Remarks:
(1813)
Time point:
24/48/72 h
Score:
1.33
Max. score:
2
Reversibility:
fully reversible within: 7 d
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Remarks:
(1815)
Time point:
24/48/72 h
Score:
1.33
Max. score:
2
Reversibility:
fully reversible within: 7 d
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #3
Remarks:
(1816)
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 72 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #4
Remarks:
(1817)
Time point:
24/48/72 h
Score:
1.33
Max. score:
2
Reversibility:
fully reversible within: 7 d
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
mean
Time point:
24/48/72 h
Score:
1.2
Max. score:
2
Reversibility:
fully reversible within: 7 d
Remarks on result:
probability of weak irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
(1813)
Time point:
24/48/72 h
Score:
0.67
Max. score:
2
Reversibility:
fully reversible within: 72 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
(1815)
Time point:
24/48/72 h
Score:
0.67
Max. score:
2
Reversibility:
fully reversible within: 72 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Remarks:
(1816)
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 7 d
Remarks on result:
probability of weak irritation
Irritation parameter:
chemosis score
Basis:
animal #4
Remarks:
(1817)
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 72 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0.8
Max. score:
2
Reversibility:
fully reversible within: 7 d
Remarks on result:
probability of weak irritation
Irritant / corrosive response data:
One hour following application of the test article all four animals exhibited a diffuse, crimson red conjunctiva with individual vessels not easily discernible with partial eversion of lids. Animal 1 (#1813) and Animal 4 (#1817) also presented a discharge and an iris with markedly deepened folds, congestion, swelling, moderate circumcorneal injection, respectively. The mean values for corneal opacity, iris lesions, redness of conjunctivae and oedema of conjunctivae (chemosis) were 0.0, 0.0, 1.2 and 0.8, respectively. All weak signs of irritation were reversible and 7 days after application of the test article all four animals were free of any signs of eye irritation.

Table 1. Scores for ocular lesions

 Ocular lesion  Animal number  1 h  24 h (after Fluorescein)  48 h

 72 h

 7 days

 Cornea opacity, degree

 1

 0

 0 (0)

 0

 0

 0
 0  2  0  0 (0)  0  0  0
   3  0  0 (0)  0  0  0
   4  0  0 (0)  0  0  0
 Cornea opacity, area  1  0  0 (0)  0  0  0
   2  0  0 (0)  0  0  0
   3  0  0 (0)  0  0  0
   4  0  0 (0)  0  0  0
 Iris  1  0  0  0  0  0
   2  0  0  0  0  0
   3  0  0  0  0
   4  1  0  0  0  0
 Conjunctiva redness  1  2  2  1  1  0
   2  2  2  1  1  0
   3  2  2  1  0  0
   4  2  2  1  1  0
 Conjunctiva chemosis  1  2  1  1  0  0
   2  2  1  1  0  0
   3  2  1  1  1  0
   4  2  2  1  0  0
 Conjunctiva discharge  1  1  0  0  0  0
   2  0  0  0  0  0
   3  0  0  0  0  0
   4  0  0  0  0  0
Interpretation of results:
GHS criteria not met
Conclusions:
The mean values for corneal opacity, iris lesions, redness of conjunctivae and oedema of conjunctivae (chemosis) were 0.0, 0.0, 1.2 and 0.8, respectively. All weak signs of irritation were reversible and 7 days after application of the test article all four animals were free of any signs of eye irritation. The test item should not be classified as irritating to the eye, according to the directive of the EEC commission 93/21/EEC and GHS/EU CLP criteria.
Executive summary:

The study was initiated with one rabbit, since no marked eye irritation was observed, another three rabbits were included. An aliquot of 0.1 mL test item was instilled into the conjunctival sac of one eye of each rabbit, while the other eye remained untreated and was used as the negative control. Assessments of damage/irritation (cornea opacity, iris lesion, conjunctival redness and conjunctival oedema) were made 1, 24, 48 and 72 hours, in addition to 7 days, following treatment.

Weak conjunctival inflammation, accompanied by slight swelling was observed in all four rabbits for up to 72 hours after instillation. The mean values for corneal opacity, iris lesions, redness of conjunctivae and oedema of conjunctivae (chemosis) were 0.0, 0.0, 1.2 and 0.8, respectively. All weak signs of irritation were reversible and 7 days after application of the test article all four animals were free of any signs of eye irritation. The test material should not be classified as irritating to the eye, according to the directive of the EEC commission 93/21/EEC (4 May 1993) and GHS/EU CLP criteria. Conducted according to OECD TG 405 (EU Method B.5) and GLP, the study was considered to be reliable (Klimisch 1).

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation: Skin irritation of the test item was evaluated with the EpiDerm™ Reconstructed Human Epidermis Model (2017). Cell viability of the multi-layered tissue culture of highly differentiated epidermal keratinocytes topically exposed to the test substance was evaluated using the MTT assay, which measures the conversion of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) into a blue formazan salt. Undiluted test item was applied to the EpiDerm™ tissue for 60 minutes, alongside a negative and positive control. The mean relative absorbance value of the test item, corresponding to the cell viability did not decrease (105.9%; threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin. The test item passed the MTT and Colour Interference pre-tests. Conducted according to OECD Test Guideline 439 and GLP, the study is considered to be reliable without restriction (Klimisch 1) and sufficient to fulfil the REACH Annex VII information requirement.

Eye irritation: Eye irritation was assessed in four female albino SPF strain rabbits with undiluted test item (2000). The study was initiated with one rabbit, since no marked eye irritation was observed, another three rabbits were included. An aliquot of 0.1 mL test item was instilled into the conjunctival sac of one eye of each rabbit, while the other eye remained untreated and was used as the negative control. Assessments of damage/irritation (cornea opacity, iris lesion, conjunctival redness and conjunctival oedema) were made 1, 24, 48 and 72 hours, in addition to 7 days, following treatment. Weak conjunctival inflammation, accompanied by slight swelling was observed in all four rabbits for up to 72 hours after instillation. The mean values for corneal opacity, iris lesions, redness of conjunctivae and oedema of conjunctivae (chemosis) were 0.0, 0.0, 1.2 and 0.8, respectively. All weak signs of irritation were reversible and 7 days after application of the test article all four animals were free of any signs of eye irritation. The test material should not be classified as irritating to the eye, according to the directive of the EEC commission 93/21/EEC (4 May 1993). Conducted according to OECD TG 405 (EU Method B.5) and GLP, the study was considered to be reliable (Klimisch 1).

Justification for classification or non-classification

Two reliable (Klimisch 1) and GLP compliant studies are available to adress the irritation / corrosion classification of the test item (2000; 2017). Whilst weak conjunctival inflammation, accompanied by slight swelling was observed in all four rabbits, the effects of the test item were reversible within 7 days. Individual mean scores were not ≥ 1 for corneal opacity and iritis and not ≥ 2 for conjunctival redness and oedema in at least 3 of 4 animals. Thus, according to the"Guidance on the Application of the CLP Criteria" (ECHA 2015), the test item should not be classified as an eye irritant. 

A substance is considered to be irritant to skin if the tissue viability after exposure and post-treatment incubation is ≤ 50%. In the EpiDerm skin irritation study according to OECD 439, the mean relative absorbance value of the test item, corresponding to the cell viability, was 105.9%. Therefore, test item is not classified as a skin irritant according to UN GHS and EU CLP Category 2.

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