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Description of key information

Three in vitro studies were conducted according to OECD guidelines 431, 437 and 439. Based on the results of these studies it can be concluded that the test substance is unlikely to be corrosive or irritating to skin nor an ocular corrosive or severe eye irritant.

Additionally, an in vivo eye irritation study was conducted according to OECD 405 and only minimal conjunctival irritation was noted. This included conjunctival redness, chemosis and discharge. All treated eyes appeared normal at the 48-Hour observation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 January 2011 to 16 August 2011
Reliability:
1 (reliable without restriction)
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD 431 In Vitro Skin Corrosion: Human Skin Model Test and Method B.40 BIS of Commission Regulation (EC) No 440/2008
Deviations:
no
GLP compliance:
yes
Species:
other: EpiDerm tissues (three-dimensional human skin model)
Details on test animals and environmental conditions:
No test animals were used.
EpiDerm tissues were supplied by MatTek Corporation, Ashland, Massachusetts, USA and stored refrigerated. Tissues were transferred to 6-well plates with the assay medium 90 minutes before starting the assay.
Type of coverage:
other: Not applicable
Preparation of test site:
other: Not applicable
Vehicle:
other: Not applicable - no vehicle used
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 40 mg
- Concentration (if solution): Undiluted test material
Duration of treatment / exposure:
Contact time of 3 minutes or 1 hour
Observation period:
Incubated at 37°C for 3 hours
Number of animals:
No animals were used on this test.
Four EpiDerm tissues were used per test substance, negative and positive control
Details on study design:
APPLICATION OF TEST AND CONTROL MATERIALS:
Immediately prior to treatment was intitiated the assay medium was replaced.
Approximately 40 mg of solid test substance was placed onto the tissue and moistened with 25 µL water to ensure good contact with the tissue surface. Further tissues were treated with 40 mL distilled water (negative control) and with 40 mL 8N potassium hydroxide (positive control).
Duplicate tissues were used for each contact time (3 minutes or 1 hour).
After the 3 minute or 1 hour contact periods, the tissues were washed with phosphate buffered saline (PBS) to remove residual material.

CELL VIABILITY MEASUREMENTS:
The assay medium was replaced with 300 mL of 1 mg/mL MTT-medium and tissues were incubated for 3 hours at 37°C. After incubation, the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol overnight, without shaking.
The optical density of the extracted formazan was determined spectrophotometrically at 570 nm and cell viability was calculated for each tissue as a percentage of the mean of the negative control tissue. Skin corrosivity potential of the test article was classified according to the remaining cell viability obtained after test article treatment with either of the two treatment times.

Irritation / corrosion parameter:
other:
Value:
105
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 3 minutes. Reversibility: other: Not measured. (migrated information)
Irritation / corrosion parameter:
other:
Value:
101
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 1 hour. Reversibility: other: Not measured. (migrated information)

Skin corrosion assay; cell viability measurements

3 minute treatment

Substance

Tissue replicate

OD540

Mean

Tissue mean

% difference between Rep’s

% Relative survival

Aliquot 1

Aliquot 2

Aliquot 3

Negative control

A

1.521

1.527

1.527

1.525

1.617

10.8

100

B

1.710

1.709

1.708

1.709

Test article

A

1.662

1.777

1.793

1.744

1.702

-5.0

105

B

1.602

1.688

1.692

1.660

Positive control

A

0.418

0.428

0.426

0.424

0.425

-0.4

26

B

0.424

0.425

0.428

0.426

        1 hour treatment

Substance

Tissue replicate

OD540

Mean

Tissue mean

% difference between Rep’s

% Relative survival

Aliquot 1

Aliquot 2

Aliquot 3

Negative control

A

1.854

1.852

1.844

1.850

1.771

-9.3

100

B

1.711

1.684

1.683

1.693

Test article

A

1.762

1.767

1.752

1.760

1.791

3.4

101

B

1.823

1.841

1.804

1.823

Positive control

A

0.224

0.238

0.228

0.230

0.239

-8.0

13

B

0.247

0.251

0.246

0.248

 

Interpretation of results:
other: Not corrosive
Remarks:
Criteria used for interpretation of results: OECD GHS
Conclusions:
The test substance was considered not to be corrosive to the invitro skin testing model, EpiDerm.
Executive summary:

The result indicates that the substance should be non-corrosive to skin. Without furtherin vivodata the result has to be considered as inconclusive regarding classification.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental dates: 25 January 2011 to 07 February 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted to GLP and in accordance with current test guidelines
Qualifier:
according to
Guideline:
other: OECD 439 In Vitro Skin Irritation Reconstructed Human Epidermis Test Method and Method B.46 of Commission Regulation (EC) No 761/2009
Deviations:
no
GLP compliance:
yes
Species:
other: EpiSkin tissues (three-dimentional human skin model)
Strain:
other: Not applicable
Details on test animals and environmental conditions:
No test animals were used.
EpiSkin tissues were supplied by SkinEthic Laboratories, Nice, France and kept in their packaging at room temperature in a microbiological safety cabinet until use. The tissues were set up 24 hours prior to treatment by placing the tissue onto a 2 mL pre-warmed maintenance medium (supplied with the EpiSkin tissues) in 12-well plates and incubating at 37°C.
Type of coverage:
other: Not applicable
Preparation of test site:
other: Not applicable
Vehicle:
other: Not applicable - no vehicle used
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 40 mg
- Concentration (if solution): Undiluted test material
Duration of treatment / exposure:
15 minutes
Observation period:
42-hour recovery period
Number of animals:
No animals were used on this test.
Three EpiSkin tissues were used per test substance, negative and positive control
Details on study design:
APPLICATION OF TEST AND CONTROL MATERIALS:
Immediately prior to treatment initiation, the media under the tissues was replaced with 2.2 mL of pre-warmed assay medium (supplied with the EpiSkin tissues).
Approximately 40 mg of the test article (moistened with 25 mL of sterile water) was added topically to the tissue. Exposure was for 15 minutes with the test article after which, the tissues were washed using PBS and dried using cotton wool buds to remove residual test material before being transferred to a new well containing 2 mL pre-warmed maintenance medium. The tissues were then incubated at 37°C for the 42 hour recovery time period.

CELL VIABILITY MEASUREMENTS:
Upon completion of the 42 hour recovery period, the base of each tissue was rinsed with PBS before being placed on top of 2 mL of 0.3 mg/mL (final concentration) MTT in medium and incubated for three hours (37°C, 5% CO2).
After incubation, the tissues were removed from the MTT solution, their bases rinsed with PBS and then placed on absorbent dry paper. The white ring was discarded and the epidermis was separated from the support layer. Both parts were placed into a sterile, sealable Eppendorf tube containing 0.5 mL acidic isopropanol. The tubes were sealed, vortexed to ensure thorough mixing and maintained at room temperature overnight protected from light.
After this extraction period, the tubes were placed in a refrigerator at 2 to 8°C for approximately 48 hours. At the end of this period, the tubes were inverted to ensure an homogenous colour. Two × 200 µL samples per tube were placed into a 96-well plate for spectrophotometric determination of optical density at 570 nm, extraction solvent was used as a blank. Tissue viability was calculated for each tissue as a percentage of the mean of the negative control tissue.

Irritation / corrosion parameter:
other:
Value:
116.7
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes exposure, 42 hour recovery period. Reversibility: other: Not measured. (migrated information)

 Skin irritatation assay; cell viability measurements

Substance

Tissue replicate

OD570(200 µL)

Corrected mean*

Standard deviation (tissue)

% Standard deviation (dose)

% Relative survival, cell viability

Aliquot 1

Aliquot 2

Tissue

Dose

Negative control

A

1.040

1.103

1.032

0.045

1.3

99.4

100.0

B

1.063

1.073

1.029

0.007

99.1

C

1.073

1.110

1.053

0.026

101.4

Test article

A

1.193

1.209

1.162

0.011

6.0

112.0

116.7

B

1.331

1.304

1.279

0.019

123.2

C

1.264

1.201

1.193

0.045

115.0

Positive control

A

0.083

0.087

0.046

0.002

3.9

4.5

7.9

B

0.107

0.124

0.077

0.012

7.4

C

0.141

0.184

0.124

0.030

11.9

Blank

0.039

0.038

 

           * = corrected for blank

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
The test substance was not considered to be an irritant to the in vitro skin testing model, EpiSkin.
Executive summary:

The result indicates that the substance should be non-irritating to skin. Without furtherin vivo data the result has to be considered as inconclusive regarding classification.

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:
From 12 June 2012 to 07 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
GLP compliance:
yes (incl. certificate)
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: 2.49 to 2.97 kg
- Housing: individually housed in suspended cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

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

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
0.1 mL of the undiluted test item, which was found to weigh approximately 62 mg
Duration of treatment / exposure:
Aftre instillation, the upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released.
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
Three male animals
Details on study design:
SCORING SYSTEM: Draize Scale for Scoring Ocular Irritation

1. CONJUNCTIVAE
(A) Redness (refers to palpebral and bulbar conjunctivae excluding cornea and iris)
Vessels normal 0
Vessels definitely injected above normal 1
More diffuse, deeper crimson red, individual vessels not easily discernible 2
Diffuse beefy red 3

(B) Chemosis
No swelling 0
Any swelling above normal (includes nictitating membrane) 1
Obvious swelling with partial eversion of lids 2
Swelling with lids about half closed 3
Swelling with lids half closed to completely closed 4

(C) Discharge
No discharge 0
Any amount different from normal (does not include small
amounts observed in inner canthus of normal animals) 1
Discharge with moistening of the lids and hairs just adjacent to lids 2
Discharge with moistening of the lids and hairs a
considerable area around the eye 3
THE TOTAL SCORE = (A + B + C) x 2 MAXIMUM TOTAL = 20


2. IRIS
(D) Values
Normal 0
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) 1
No reaction to light, haemorrhage, gross destruction (any or all of these) 2
THE TOTAL SCORE = D x 5 MAXIMUM TOTAL = 10

3. CORNEA
(E) Degree of Opacity (most dense area used)
No opacity 0
Scattered or diffuse areas, details of iris clearly visible 1
Easily discernible translucent areas, details of iris slightly obscured 2
Opalescent areas, no details of iris visible, size of pupil barely discernible 3
Opaque, iris not discernible through the opacity 4

(F) Area of Cornea Involved
One quarter (or less) but not zero 1
Greater than one quarter but less than half 2
Greater than half but less than three quarters 3
Greater than three quarters, up to whole area 4
THE TOTAL SCORE = (E x F) x 5 MAXIMUM TOTAL = 80

MAXIMUM TOTAL SCORE POSSIBLE = 110


The 24h examination was performed under ultra-violet light following treatment with Sodium Fluorescein BP
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
all three animals
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
mean
Remarks:
all three animals
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
animal: all three animals
Time point:
24/48/72 h
Score:
0.33
Max. score:
1
Reversibility:
fully reversible
Irritation parameter:
chemosis score
Basis:
other: all three animals
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritant / corrosive response data:
Slight redness of the conjunctivae was noted in all animals at the 1h- and 24h- reading.
Slight chemosis was also noted in all animals at the 1h-reading in all animals and slight discharge was noted at the same observation period in two animals.
All reactions were fully reversible and the animals were clean after 48 hours.

The body weight of the animals were within the range for animals of this strain and age.

Interpretation of results:
GHS criteria not met
Conclusions:
The substance is not considered to be irritant for eye according the GHS criteria.
Executive summary:

An in vivo eye irritation study was conducted with three male adult New Zealand rabbits according to OECD guideline 405.

0.1 mL of the test substance was instilled in the left eye of each animal and the animals were scored approximately 1, 24, 48 and 72 hours after instillation.

After single application of the test item to the non-irrigated eye of the three rabbits only minimal conjunctival irritation was noted. This included conjunctival redness, chemosis and discharge. All treated eyes appeared normal at the 48-Hour observation.

The test item is not considered to be irritant for the eyes according to GHS criteria.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the above mentioned studies, the substance does not have to be classified for skin or eye irritation accordingto CLP (Regulation (EC) No 1272/2008 Of the European parliament and of the Council.