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Description of key information

Skin irritation. Key study. Method according to OECD guideline 439, GLP study. The test item is not irritant since the mean percent viability of the treated tissues was 100.19% in the RHE test.

Eye irritation. Key study. Method according to OECD guideline 437, GLP study. The test item cannot be predicted as not classified for eye irritation/serious eye damage or as causing serious eye damage with the BCOP test method.

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:
23 February 2018 - 01 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Test system:
human skin model
Remarks:
EpiSkinTM
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Source strain:
not specified
Justification for test system used:
The assessment of skin irritation has typically involved the use of laboratory animals (OECD TG 404). In relation to animal welfare concerns, TG 404 recommends the use of a tiered testing strategy for the determination of skin corrosion and irritation which includes the use of validated in vitro or ex vivo test methods avoiding pain and suffering of animals.
One of the validated in vitro test methods adopted by the OECD TG 439 makes use of reconstructed human epidermis (RhE) which closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM kit
- Tissue batch number(s): 18-EKIN-009
- Delivery date: 27 February 2018
- Expiration date: 05 March 2018
- Date of initiation of testing: 23 February 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: PBS, volumen no specified
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours at 37°C, 5% CO2
- Spectrophotometer: TECAN Infinite® M200 Microplate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Historical negative control mean OD range = 0.58630-1.13620 (15 min exposure).
- Histology scoring: 21.5 ± 1.2 (CV = 5.7%), specification ≥ 19.5.
- Barrier function: IC50 = 2.1 mg/mL (specification, 1.5 mg/mL ≤ IC50 ≤ 3 mg/mL)
- Morphology: Well-differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: no interference.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability after after exposure and post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the tissue viability after exposure and post-treatment incubation is greater than 50%.


Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 μL (26 μL/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% SDS
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
100.19
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Remarks:
(PBS)
Positive controls valid:
yes
Remarks:
6.87% viability (5% SDS)
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A demonstration of proficiency was performed for the EpiSkinTM kit. Adequate results were obtained for the evaluated chemicals.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, OD mean is 0.69413 (acceptability criteria, 0.6≤OD≤1.5). SD of the negative control group was 0.00378 (acceptablility criteria, SD ≤ 18%).
- Acceptance criteria met for positive control: yes, SD of the positive control group was 0.21% (acceptablility criteria, SD ≤ 18%)
- Acceptance criteria met for variability between replicate measurements: yes. SD of test item was 1.23% (acceptablility criteria, SD ≤ 18%).

Table 1. Mean OD Values of Individual Epidermis Units

 

Absorption (OD570)

 

R1

R2

R3

Mean

Negative control

0.73520

0.73360

0.74080

 

0.73653

Positive control

0.09110

0.09080

0.08845

 

0.09012

Test Item

0.74580

0.73885

0.72885

 

0.73783

Table 2. True OD Values of Individual Epidermis Units

 

Absorption (OD570)

R1

R2

R3

Mean

SD

Negative control

0.69280

0.69120

0.69840

0.69413

0.00378

Positive control

0.04870

0.04840

0.04605

0.04772

0.00145

Test Item

0.70340

0.69645

0.68645

0.69543

0.00852

Blank OD Value (mean of 6 replicate values): 0.04240

True OD value = OD Raw – OD Blank

Table 3. Individual Tissue Viability of Epidermis Units (Relative)

 

% Individual Viability

R1

R2

R3

Mean

SD

Positive control

7.02

6.97

6.63

6.87

0.21

Test Item

101.33

100.33

98.89

100.19

1.23

Interpretation of results:
other: No category (CLP Regulation EC no. 1272/2008)
Conclusions:
Under in vitro RHE test method performed in Episkin model the test item is predicted to be non-irritant.

Executive summary:

An in vitro skin irritation test of the test item was performed in a reconstructed human EpiSkin TM model, according to OECD TG 439 (GLP study). Three epidermis units were treated with 10 μL test item for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with PBS. The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol, incubating the tissues at room temperature protected from light for 4 hours, and finally measuring the concentration of formazan by determining the OD at 570 nm. Under the test conditions, the mean percent viability of the treated tissues was 100.19%, versus 6.87% of the positive control (5% Sodium Dodecyl Sulfate) and 100% of the negative control (PBS). Therefore, the test item is predicted to be non-irritant.

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:
20 February 2018- 25 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Local abattoir (Slaughter house), Near Frazer town, Bengaluru.
- Characteristics of donor animals (e.g. age, sex, weight): not specified.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The bovine eyes were procured from local abattoir and transported in a jar containing Hank’s Balanced Salt Solution (HBSS) and penicillin-streptomycin (100 IU & 100 µg/mL) in an ice box.
- Time interval prior to initiating testing: Not specified.
- indication of any existing defects or lesions in ocular tissue samples: immediately after receiving the eyes in the lab, all eyes were observed for any evidence of vascularization, pigmentation, opacity or scratches. Corneas from eyes free of visible defects were used. 2 out of 12 corneas exhibited initial opacity more than 7. The corneas which exhibited initial opacity < 7 were considered for further experiment.
- Indication of any antibiotics used: yes, penicillin at 100 IU/mL and streptomycin at 100 μg/mL.
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): 750 μL
- Concentration (if solution): undiluted

Duration of treatment / exposure:
10 min
Duration of post- treatment incubation (in vitro):
2 h
Number of animals or in vitro replicates:
3 replicates.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: Immediately after receiving the eyes in the lab, all eyes were observed for any evidence of vascularization, pigmentation, opacity or scratches. Post pretest examination, selected eyes were dissected to isolate the corneas from surrounding tissue and then placed in a container with fresh HBSS (Hank’s Balanced Salt Solution). The isolated corneas were mounted on the cornea holder. During mounting, it was ensured that the epithelium of the cornea projected into the anterior chamber. EMEM (Eagle Minimum Essential Medium) without phenol red was added in to both the chambers and kept in an incubator (32°C) for 1 hour and 15 minutes. After pre-test incubation, the EMEM from both the chambers was removed and refilled with the fresh EMEM. The pre-treatment cornea reading (lux) was measured and initial opacity was calculated for all corneas. 2 out of 12 corneas exhibited initial opacity more than 7. The corneas which exhibited initial opacity < 7 were considered for further experiment. Post initial opacity measurement, nine corneas with opacity less than 7 were grouped into Negative control, Positive control and Test item treatment groups with 3 corneas per group.

QUALITY CHECK OF THE ISOLATED CORNEAS: Corneas from eyes free of visible defects and with an opacity value inferior than 7 were used.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: yes. Distilled water.

POSITIVE CONTROL USED: yes. Acetone.

APPLICATION DOSE AND EXPOSURE TIME: 750 μL of undiluted test item, 10 min exposure.

TREATMENT METHOD: closed chamber.

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Anterior chamber was washed three times with EMEM containing phenol red. After, the anterior chamber was rinsed with EMEM without phenol red.

- POST-EXPOSURE INCUBATION: After rinsing, both the chambers were filled with fresh EMEM and further incubated at 32 (±1) ºC for 2 hours before measuring the final opacity.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Final opacity of all corneas was measured with fresh EMEM using an OP3.0 Opacitometer (Duratec, Germany)
- Corneal permeability: passage of sodium fluorescein dye (optical density at 490 nm) measured with microplate reader (Tecan, Model: Infinite M200).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: as indicated in the TG (see table below).

Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
18.045
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Remarks:
1.429
Positive controls valid:
yes
Remarks:
104.32
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
18
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Remarks:
1.33
Positive controls valid:
yes
Remarks:
96.34
Irritation parameter:
other: permeability
Run / experiment:
mean
Value:
0
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Remarks:
0.0066
Positive controls valid:
yes
Remarks:
0.5320
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- A test is considered acceptable if: the positive control gives an IVIS that falls within two standard deviations of the current historical mean and negative/solvent controls gives values of opacity and permeability lower than upper limits for background values.
- Acceptance criteria met for negative control:yes, distilled water response resulted in opacity and permeability values that are less than established upper limits for negative control opacity (1.33) and permeability values (0.0257)
- Acceptance criteria met for positive control: yes, IVIS score of acetone was 104.32 which falls between two standard deviations of the historical mean i.e. 96-128.

Table 1. Details of Opacity Calculation.

Cornea

Holder Number

Blank value*

Io (lux)

Pre-treatment Cornea reading#

I (lux)

Initial opacity

(t0)*

Treatment group

Post-treatment Cornea reading$

I (lux)

Final opacity

(10 mins)*

Change of Opacity

 

 

Corrected Opacity

 

 

Mean Opacity

2016-123#01

502

453

5

Negative control: Distilled water

448

5

0

NA

1.33

2016-123#02

502

482

2

454

5

3

2016-123#04

502

472

3

458

4

1

2016-123#21

502

451

5

Positive control: Acetone

140

103

98

96.67

96.34

2016-123#22

502

431

7

130

114

107

105.67

2016-123#23

502

472

3

153

91

88

86.67

2016-123#11

502

433

7

Test item: 3,3-Dimethyl-8,9-Dinorbornan-2-one

319

23

16

14.67

18.00

2016-123#13

502

444

6

346

18

12

10.67

2016-123#14

502

449

5

268

35

30

28.67

Remarks:

* Blank value for each cornea holder with medium but without cornea.

# Reading of each cornea holder with cornea and medium (Post pre-test incubation period).

$ 10 minutes post reference item exposure incubation period

Initial/Final opacity = ((I0/I)-b)/a, where I = individual reading, a=0.0251 & b=0.9894 (a and b values are instrument specific constants).

Change of opacity = Final opacity – Initial opacity

Corrected opacity = Change in opacity of each treated cornea – average change of opacity value of negative corneas i.e. “1.33” of distilled water

Mean opacity = Mean of corrected opacity

Table 2. Details of Permeability Calculation

Cornea

Holder Number

Treatment group

OD490reading

(90 mins)

Blank corrected OD490*

Negative control corrected OD490#

Mean

 

 

NA

 

Blank OD reading

 

0.0387

NA

 

 

NA

0.0390

0.0379

0.0385

0.0391

0.0414

0.0382

 

 

2016-123#01

Negative control: Distilled water

0.0471

0.0081

 

NA

0.0066

0.0479

0.0089

2016-123#02

0.044

0.0050

0.046

0.0070

2016-123#04

0.0444

0.0054

0.0442

0.0052

 

 

2016-123#21

Positive Control:

Acetone

0.5713

0.5323

0.4867

0.5320

0.5627

0.5237

0.4781

2016-123#22

0.3654

0.3264

0.2808

0.3687

0.3297

0.2841

2016-123#23

0.9224

0.8834

0.8378

0.9088

0.8698

0.8242

 

2016-123#11

Test item: 3,3-Dimethyl-8,9-Dinorbornan-2-one

0.0765

0.0375

-0.0081

0.0003

0.075

0.0360

-0.0096

2016-123#13

0.0965

0.0575

0.0119

0.0975

0.0585

0.0129

2016-123#14

0.0807

0.0417

-0.0039

0.0833

0.0443

-0.0013

Remarks:

* Blank corrected OD490 = Individual corneal reading OD490 – Mean OD Blank reading

# Negative control corrected OD490 = Individual blank corrected OD490 – Mean of Negative control OD490 reading (Distilled water)

Table 3. Calculation of In Vitro Irritation Score (IVIS*)

Treatment group

Mean Opacity value

Mean Permeability value

IVIS

Classification

Negative control

(Distilled water)

1.33

0.0066

1.429

No Category

Acetone

96.34

0.5320

104.32

Category I

3,3-Dimethyl-8,9-Dinorbornan-2-one

18

0.0003

18.045

No Prediction can be made

*IVIS = Mean Opacity value + (15 × Mean Permeability value)

Interpretation of results:
other: No prediction can be made (CLP Regulation EC no. 1272/2008)
Conclusions:
The test item cannot be predicted as not classified for eye irritation/serious eye damage or as causing serious eye damage with the BCOP test method.

Executive summary:

An in vitro Bovine Corneal Opacity and Permeability (BCOP) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 437 under GLP conditions. Three sets consisting of three corneas each were tested: the first set was the negative control, and was treated with 750 μL distilled water; the second set was the positive control, and was treated with 750 μL of acetone and the third set was treated with 750 μL of test item. The corneas were exposured for 10 min after which the test item was washed and then the corneas were kept in incubation for 2 h at 32ºC. After incubation, opacity of the corneas and fluorescein permeability were measured. Negative control and positive control exhibited expected response as No category (IVIS score 1.429) and Category 1 (IVIS score 104.32) respectively. The test item exhibited an IVIS score of 18.045. Thus, it can be concluded that the test ítem cannot be predicted as not classified for eye irritation/serious eye damage or as causing serious eye damage with the BCOP test method.

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation. Key study. An in vitro skin irritation test of the test item was performed in a reconstructed human EpiSkin TM model, according to OECD TG 439 (GLP study). Three epidermis units were treated with 10 μL test item for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with PBS. The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol, incubating the tissues at room temperature protected from light for 4 hours, and finally measuring the concentration of formazan by determining the OD at 570 nm. Under the test conditions, the mean percent viability of the treated tissues was 100.19%, versus 6.87% of the positive control (5% Sodium Dodecyl Sulfate) and 100% of the negative control (PBS). Therefore, the test item is predicted to be non-irritant.

Eye irritation. Key study. An in vitro Bovine Corneal Opacity and Permeability (BCOP) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 437 under GLP conditions. Three sets consisting of three corneas each were tested: the first set was the negative control, and was treated with 750 μL distilled water; the second set was the positive control, and was treated with 750 μL of acetone and the third set was treated with 750 μL of test item. The corneas were exposured for 10 min after which the test item was washed and then the corneas were kept in incubation for 2 h at 32ºC. After incubation, opacity of the corneas and fluorescein permeability were measured. Negative control and positive control exhibited expected response as No category (IVIS score 1.429) and Category 1 (IVIS score 104.32) respectively. The test item exhibited an IVIS score of 18.045. Thus, it can be concluded that the test ítem cannot be predicted as not classified for eye irritation/serious eye damage or as causing serious eye damage with the BCOP test method.

Justification for classification or non-classification

Based on the available information, the test item is not classified for skin irritation/corrosion in accordance with CLP Regulation (EU) No. 1272/2008.

Based on the available information, the test item cannot be predicted as not classified for eye irritation/serious eye damage or as causing serious eye damage in accordance with CLP Regulation (EU) No. 1272/2008.