Registration Dossier

Administrative data

Description of key information

Skin irritation/corrosion: Not irritating (OECD 439, GLP, K, rel. 1).

Eye irritation: Irritating, top-down approach:

- ICE: no prediction can be made (OECD 438, GLP, WoE, rel.1)

- EpiOcular: potentially requiring classification and labelling according to the EU CLP (Category 1 or 2) (OECD 492, GLP, WoE, rel.1)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 November 2015 - 21 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 439 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 17 June 2015 / signed on 24 September 2015)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Following the REACH bottom-up strategy, the EPISKIN™ Reconstructed Human Epidermis Model method was used to assess skin irritation as recommended in the OECD test guideline No. 439.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 15-EKIN-050
- Production date: not reported
- Shipping date: 15 December 2015
- Delivery date: 15 December 2015
- Expiry date: 21 Decemer 2015
- Date of initiation of testing: 16 Decembre 2015

Prior to dosing the test item was heated to 45 oC to aid handling.

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: not reported
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in DPBS
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm (without a reference filter)
- Filter: not applicable
- Filter bandwidth: not applicable
- Linear OD range of spectrophotometer: no data reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: negative control OD values: 0.765, 0.626 and 0.736 (historical control: mean OD = 0.808; range 0.626 to 1.245).
- Barrier function: IC50= 2.3 mg/mL ( ≥ 1.5 mg/mL)
- Morphology: well differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: : absence of bacteria, fungus and mycoplasma
- Reproducibility: not reported

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not applicable

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 viability after the 15-min exposure period followed by the 42h post-exposure period is less or equal to 50%
- The test substance is considered to be non-irritant to skin if the viability after the 15-min exposure period followed by the 42h post-exposure period 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): Approximately 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal surface.
- Concentration (if solution): not applicable

VEHICLE
not applicable

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL of DPBS
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL of SDS
- Concentration (if solution): 5% w/v aqueous solution



Duration of treatment / exposure:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes at room temperature.
At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5% CO2 in air for 42 h.
Duration of post-treatment incubation (if applicable):
- On Day 3, at the end of the 42 h post-treatment incubation period: MTT test (MTT Loading/Formazan Extraction) was performed and tissues were incubated for 3 h at 37 °C, 5 % CO2 in air.
- On Day 6, at the end of the formazan extraction period: The optical density was measured at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.
Number of replicates:
Triplicate tissues for test item, negative and positive controls
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 minute exposure period and 42 h post-exposure incubation period.
Value:
101.8
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:
The relative mean viability of the test item treated tissues was 101.8% (calculated relative to the negative control group) after a 15 minute exposure period and 42 h post-exposure incubation period. It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: For the previous 27 experiments conducted between 05 May 2015 and 21 October 2015 up to the start of this study (approximately 6 months data) using this test method, the mean OD of the positive control was 0.089; range 0.049 to 0.170 and the mean percentage viability was 11.3; range 6.2 to 21.5. In this same period the mean OD of the negative control was 0.808; range 0.629 to 1.245.
It was considered that comparison of the historical control data supports correct functioning of the test system. The results of this study are therefore considered acceptable.

Table 7.3.1/1: Mean OD562Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

 

Item

OD562 of tissues

Mean OD562 of triplicate tissues

± SD of OD562

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.765

0.709

0.073

107.9

100*

10.3

0.626

88.3

0.736

103.8

Positive Control Item

0.180

0.040

0.040

25.4

18.9

5.6

0.112

15.8

0.110

15.5

Test Item

0.700

0.722

0.019

98.7

101.8

2.7

0.732

103.2

0.733

103.4

SD=        Standard deviation

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

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 18.9% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 5.6%. The positive control acceptance criterion was therefore satisfied.

The mean OD562 for the negative control treated tissues was 0.709 and the standard deviation value of the percentage viability was 10.3%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically test item treated tissues were 2.7%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test item is not classified according to Regulation (EC) No. 1272/2008 (CLP).
Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model.

This study was run twice due to a failed Quality Control result in the first run. The report is based on the results of the study generated in the acceptable second run. 

 

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 h. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 562 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

The relative mean viability of the test item treated tissues was 101.8% after the 15 minute exposure period and 42 h post-exposure incubation period.

 

The relative mean tissue viability for the positive control treated tissues was 18.9% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 5.6%. The mean OD562 for the negative control treated tissues was 0.709 and the standard deviation value of the percentage viability was 10.3%. The positive and negative controls acceptance criterion were therefore satisfied. The standard deviation calculated from individual percentage tissue viabilities of the three identically test item treated tissues were 2.7%. The test item acceptance criterion was therefore satisfied.

 

Under the experimental conditions of this study, the test item is not classified according to Regulation (EC) No. 1272/2008 (CLP).

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
11 January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 438 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye 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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected on 17 June 2015 / signed on 24 September 2015
Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Multiple chicken heads, were supplied by Baileys Turkeys Ltd., Cheshire, UK where they were killed for human consumption were used for this assay.
- Characteristics of donor animals (e.g. age, sex, weight): Spring chickens (Gallus Gallus e.g. Ross 308 Broiler) weighed 3kg and were approximately 56 days old.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day. The time interval between collection of chicken heads (9:55am- 11 January 2016) and placing the eyes in the superfusion chamber (10:29am- 11 January 2016) following enucleation was minimized although all eyes had to fall within the acceptance criteria identified in the test guideline. The temperature of the chambers was at 32 ±1.5 °C.
Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.
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): 30 µL of the test item was applied, as supplied, to the cornea
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
The test item was applied for 10 seconds and then rinsed from the eye using 20mL of isotonic saline
Number of animals or in vitro replicates:
Test item: 3 eyes
Positive control: 3 eyes
Negative control: 2 eyes
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
- Eyelids were carefully excised whilst taking care not to damage the cornea. The integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined with the use of the Haag-Streit BQ 900 (Switzerland) microscope, to examine for damage to the cornea. An acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores are =< 0.5.
- Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt tipped scissors. Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner.
- Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32 ±1.5 °C.
- Once all eyes were placed in the superfusion apparatus, the eyes were examined again with the Haag-Streit BQ 900 to ensure the eyes had not been damaged by the dissection procedure. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit lamp microscope at the center of each cornea.
- Eyes were replaced when: (i) the fluorescein score was > 0.5; (ii) the corneal opacity score was > 0.5; or (iii) there was any additional signs of damage, (iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes.
- After the approval process the eyes were incubated for 45 minutes for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

NUMBER OF REPLICATES: 2, 3 and 3 eyes for negative & positive control and test item, respectively.

NEGATIVE CONTROL USED: sodium chloride 0.9% w/v

POSITIVE CONTROL USED: 5% Benzalkonium chloride

APPLICATION DOSE AND EXPOSURE TIME
- Immediately following the zero reference measurements, each test eye (including clamp) was removed from the superfusion apparatus and placed horizontally (cornea facing upwards) into a petri dish. 0.03 mL of test item was applied to the cornea. The entire surface of the cornea was evenly covered. The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of isotonic saline. The treated eye (including clamp) was subsequently returned to the superfusion apparatus in the original upright position.

OBSERVATION PERIOD
- Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the eyes had been decontaminated with the isotonic saline.

METHODS FOR MEASURED ENDPOINTS:
- All observations of the cornea and measurement of corneal thickness were performed using a HaagStreit BP900 slit-lamp microscope with depth-measuring device no. 1.
- Endpoints used during the evaluation procedure were corneal opacity, swelling, fluorescein retention and morphological effects (e.g. pitting, sloughing or roughening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which is only determined at 30 minutes after test substance exposure) were determined at each of the above time points.

SCORING SYSTEM:
- Mean corneal swelling (%): Percentage corneal swelling was assessed from corneal thickness measurements. Mean percentage of corneal swelling for all test eyes was calculated for all the time points. The overall category score was determined from the highest mean score for epithelial swelling as observed at any time point.
Corneal swelling (%) = ((corneal thickness at time t - corneal thickness at time = 0) / (corneal thickness at time = 0)) x 100
- Mean maximum opacity score: Corneal opacity was calculated with the most densely opacified areas for scoring. The mean value for all test eyes was calculated for all time points. The highest mean score, as observed at any time point was given an overall category for each test item.
0: No opacity
0.5: Very faint opacity
1: Scattered or diffuse areas; details of the iris clearly visible
2: Easily discernible translucent area; details of the iris are slightly obscured
3: Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4: Complete corneal opacity; iris invisible
- Mean fluorescein retention score at 30 minutes post-treatment
0: No fluorescein retention
0.5: Very minor single cell staining
1: Single cell staining scattered throughout the treated area of the cornea
2: Focal or confluent dense single cell staining
3: Confluent large areas of the cornea retaining fluorescein
- Pitting, loosening (sloughing), roughening of the corneal surface, and adhering of test item are all morphological effects that can be noted on the cornea. The classification of these findings was subject to interpretation.

DECISION CRITERIA:
Results from corneal opacity, swelling, and fluorescein retention should be evaluated separately to generate an ICE class for each endpoint. The ICE classes for each endpoint are then combined to generate an Irritancy Classification for each test item.
Irritation parameter:
cornea opacity score
Value:
1.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Value:
20.79
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
The ocular reactions observed in eyes treated with the test item were:
- maximal mean score of corneal opacity: 1.3, corresponding to the ICE class II;
- mean score of fluorescein retention: 1.0, corresponding to the ICE class II;
- maximal mean corneal swelling: 20.79% corresponding to the ICE class III.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The combination of the three endpoints for the negative control, physiological saline solution, was 2 x I, 1 x II. Therefore, the negative control is classified as "No Category", as expected.
- Acceptance criteria met for positive control: The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe irritant", as expected.

Table 7.3.2/5: Individual and average values for evaluation of corneal lesions after treatment with the test item

 

End Point

Eye Number

Time
(after decontamination)

0
minutes

30 minutes

75 minutes

120 minutes

180 minutes

240 minutes

Corneal Opacity

3A

0

0.5

2

2

2

2

6A

0

0.5

1

1

1

1

8A

0.5

0.5

0.5

0.5

1

1

Mean

0.2

0.5

1.2

1.2

1.3

1.3

ICE Class

II

Fluorescein Retention

3A

 

1

 

 

 

 

6A

 

2

 

 

 

 

8A

 

0

 

 

 

 

Mean

 

1.0

 

 

 

 

ICE Class

II

Corneal Thickness

3A

0.64

0.72

0.80

0.82

0.84

0.84

6A

0.64

0.72

0.66

0.72

0.76

0.76

8A

0.74

0.78

0.80

0.80

0.80

0.84

Mean

0.67

0.74

0.75

0.78

0.80

0.81

Mean Corneal Swelling (%)

 

9.90

11.88

15.84

18.81

20.79

ICE Class

III

ICE Classes Combined:

II, II, III

Classification:

No prediction of eye irritation can be made

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the test conditions, no prediction can be made according to the Annex VI of Regulation (EC) No. 1272/2008 (CLP).
Executive summary:

An in vitro eye irritation study was performed according to the OECD Guideline 438 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

 

The test item was applied, as supplied, at the dose of 30 µL, to 3 enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed using 20 mL of isotonic saline. Three eyes were treated in the same manner with a positive control and two eyes with a negative control. Damages by the test substance were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 1.3, corresponding to the ICE class II;

- mean score of fluorescein retention: 1.0, corresponding to the ICE class II;

- maximal mean corneal swelling: 20.79% corresponding to the ICE class III.

 

The combination of the three endpoints for the negative control, physiological saline solution, was 2 x I, 1 x II. Therefore, the negative control is classified as "No Category", as expected. The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe irritant", as expected.

 

Under the test conditions, no prediction can be made according to the Annex VI of Regulation (EC) No. 1272/2008 (CLP).

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
18 October to 03 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 492 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
July 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model
Version / remarks:
29 June 2015
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected from 2015-07-13 to 2015-07-16 / signed on 2015-09-14
Species:
other: human reconstructed cornea model (EpiOcular™)
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. This test guideline is applicable to solid and waxes, so is considered to be applicable to the test item.

CELL SYSTEM:
- EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (Ashland, MA01721, USA).
- Lot No.: 21744
- Keratinocyte strain: 4F1188
- Analysis for potential biological contaminants: none detected (HIV-1, Hep. B and Hep. C virus; Bacteria, yeast and other fungi)
- Tissue viability: 1.591, within the acceptance criteria (1.1-3.0)
- Barrier function: 27.8 min, within the acceptance criteria (12.2-37.5)
- Sterility: Sterile

- The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELL, 10 mm of diameter).
- Transport: EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate.
- Storage: On day of receipt of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. An appropriate volume of EpiOcular™ Assay Medium was warmed to approximately 37 °C. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.
Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (nearly 17 hours).
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): 50 mg (83.3 mg/cm²)
Duration of treatment / exposure:
6 hours
Observation period (in vivo):
NA
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
duplicate tissues
Details on study design:
- Details of the test procedure used: procedure for solids
- Doses of test chemical and control substances used: 50 mg
- Duration and temperature of:
exposure: 6 hours / 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH
post-exposure immersion: 25 min / room temperature
post-exposure incubation period: 18 hours / 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH
- Description of any modifications to the test procedure:
Assessment of Coloured or Staining Materials: Since the test item was brown-red and dyed and isopropanol in the colour interference pre-test, it was relinquished to determine the absorption at 570 nm, and it was decided to perform an additional test with viable tissues (without MTT addition).
- Indication of controls used for colouring test chemicals: Since the test item’s colour was red-brown, and since it dyed isopropanol (yellow), it was relinquished to determine the absorption at 570 nm, and the additional test with viable tissues (without MTT addition) was performed to determine a correction factor for calculating the true viability.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving): 2
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 ml of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Inserts were removed from the 24-well plate after 180 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 6-well plate containing 2 ml isopropanol in each well so that no isopropanol was flowing into the insert. The corresponding negative and positive controls had to be treated identically without piercing. For this procedure it was necessary to seal the plates particularly thorough since a higher evaporation rate had to be expected due to the larger surface of wells in 6¬-well plates.
Extraction was performed for about 2.2 hours a room temperature on a shaker.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate(s).
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1). No reference wavelength measurement was used.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The results are acceptable according to OECD TG 492 if:
1) The negative control OD is > 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 50% of the negative control viability.
3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the freeze-killed tissues (items and negative control) and the additional viable tissues (without MTT addition) which are calculated as percent values related to the viability of the relating negative control.
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is identified potentially requiring classification and labelling according
to UN GHS (Category 2 of Category 1.
According to OECD guideline 492 a single test composed of at least two tissue replicates should be sufficient for a test chemical, when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent tissue viability equal to 60±5%, a second test should be considered, as well as a third one in case of discordant results between the first two tests.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Data of 11 studies performed from July 2015 until end of February 2016
Positive controls: Viability 32.0% (6.91-40.4) / Absorption 0.538 (0.107-0.849)
Negative controls: Absorption 1.65 (1.27-2.05)
- Complete supporting information for the specific RhCE tissue construct used: yes, attached to the study report
- Reference to historical data of the RhCE tissue construct: no
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: yes, attached to the study report
- Positive and negative control means and acceptance ranges based on historical data: yes
- Acceptable variability between tissue replicates for positive and negative controls: yes
- Acceptable variability between tissue replicates for the test chemical: yes
Irritation parameter:
other: tissue viability
Run / experiment:
Mean
Value:
32.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The negative control OD is > 0.8 and < 2.5 (values between 1.582 and 1.771).
- Acceptance criteria met for positive control: yes. The mean relative viability of the positive control is below 50% of the negative control viability (37.0%).
Acceptable variability between tissue replicates: yes. The difference of viability between the two relating tissues of a single item is < 20% (values between 0.1% to 8.2%) in the same run. This does also apply to the additional viable tissues (without MTT addition).
Irritant / corrosive response data:
Negative control: Tissue viability = 100%.
Positive control:Tissue viability = 37 %.
Test item: Tissue viability = 33.3 % (32.2% if corrected)
Other effects:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol led to a change in colour of isopropanol. Also the test item’s colour was intensive (dark brown). Therefore, an additional test with viable tissues without MTT addition was necessary to determine a correction factor for calculating the true viability in the main experiment.
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue/purple colour. Therefore, an additional test with freeze-killed tissues was not necessary.

Table 7.3.2/1: Results after 6 hour treatment with Resinoid of Commiphora myrrha (Burseraceae) obtained from the gum by ethanol extraction and the controls

Dose Group

Ab-sorbance (OD) 570 nm
Well 1

Ab-sorbance (OD) 570 nm
Well 2

Mean Ab-sorbance (OD) of 2 Wells minus Blank

Mean Ab-sorbance (OD) of 2 Tissues

Mean Absorbance [%]

Mean Rel. Absorbance
[% of Negative Control]*

Absolute Value of the Difference of the Rel. Absorbances [%]
Tissue 1 and 2

Corrected Rel. Absorbance [% of Negative Control]**

Blank

0.039

0.040

0.000

 

 

 

 

 

Negative Control Tissue 1

1.682

1.771

1.688

1.621

104.1

100.0

8.2

 

Negative Control Tissue 2

1.604

1.582

1.554

95.9

Positive Control Tissue 1

0.647

0.672

0.621

0.600

38.3

37.0

2.6

Positive Control Tissue 2

0.617

0.619

0.579

35.7

Test Item Tissue 1

0.572

0.599

0.546

0.539

33.7

33.3

0.8

32.2

Test Item Tissue 2

0.573

0.570

0.533

32.9

Negative ControlAdd.Viable TissueTissue 1

0.042

0.042

0.003

0.005

0.2

0.3

0.1

 

Negative ControlAdd. Viable TissueTissue 2

0.046

0.045

0.006

0.4

Test ItemAdd. Viable TissueTissue 1

0.054

0.056

0.016

0.017

1.0

1.0

0.1

Test ItemAdd. Viable TissueTissue 2

0.057

0.058

0.018

1.1

Interpretation of results:
study cannot be used for classification
Conclusions:
With a mean percent tissue viability < 60%, the test item potentially require classification and labelling according to EU CLP (Category 2 or Category 1).
Executive summary:

A study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test. The study was conducted according to the OECD guideline No. 492 and in compliance with GLP.

The test item did not prove to be an MTT reducer in the MTT pre-test, therefore, an additional test with freeze-killed tissues was not performed. Its intrinsic colour was intensive and it proved to dye isopropanol (yellow) in the colour interference pre-test. Therefore, an additional test with viable tissues (without MTT addition) was performed to determine a correction factor for calculating the true viability in the main experiment.

50 mg of the test item was applied to each of duplicate tissue for 6 hours. Each 50 µL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD > 0.8 and < 2.5 thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 50% compared with thenegative control value in the relative absorbance thus ensuring the validity of the test system.

The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

Irritating effects were observed following incubation with the test item. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (33.3%, if the correction factor derived from the additional test with viable tissues was not taking into account; 32.2%, if corrected).

In conclusion, with a mean percent tissue viability < 60%, the test item potentially require classification and labelling according to EU CLP (Category 2 or Category 1).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

Since no key study was identified on the registered substance, the testing and assessment strategy, as described in ECHA R.7a Endpoint specific guidance (December 2016), was used to evaluate the skin corrosion/irritation potential of the registered substance:

 

Element

Information

Conclusion

Comments

Existing data on physico
- chemical properties

1a

Is the substance spontaneously flammable in contact with air (pyrophoric) or water at room temperature?

NO

 

1b

Is the substance an organic hydroperoxide or an organic peroxide?

NO

 

1c

Is the pH of the substance ≤ 2.0 or ≥ 11.5?

NO

 

1d

Are there other physical or chemical properties that
indicate that the substance is corrosive/irritant?

NO

 

Existing human data

2

Are there adequate existing human data which provide evidence that the substance is a corrosive
or irritant?

NO

 

Existing animal data from corrosion/irritation studies

3

Are there data from existing studies on corrosion and irritation in laboratory animals, which provide sound conclusive evidence that the substance is a corrosive, irritant or non-irritant?

NO

 

Existing data from general toxicity studies via the dermal route and from sensitisation studies

4a

Is the substance classified as fatal in contact with skin (LD50 ≤ 50 mg/kg bw, CLP hazard statement
H310)

NO

 

4b

Has the substance proven to be a corrosive, irritant or non-irritant in a suitable acute dermal toxicity test?

NO

 

4c

Has the substance proven to be a corrosive or an irritant in sensitisation studies or after repeated
exposure?

NO

 

Existing/new (Q)SAR data and read
-across

5a

Are there structurally related substances (suitable “read-across” or grouping), which are classified as corrosive to the skin (Skin Corrosive Cat. 1), or do suitable (Q)SAR methods indicate corrosion
potential of the substance?

NO

Not applicable - UVCB substance

5b

Are there structurally related substances (suitable “read-across” or grouping), which are classified as irritant to the skin (Skin Irritant Cat. 2), or indicating that the substance is non-irritant, or do suitable (Q)SAR methods indicate irritant or non-irritant potential of the substance?

NO

 

Existing in vitro data

6a

Has the substance demonstrated corrosive properties in an EU/OECD adopted in vitro test?
Data from in vitro test methods that have been validated and are considered scientifically valid but
are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met

NO

 

6b

Has the substance demonstrated irritant or non-irritant properties in an EU/OECD adopted in vitro test?
Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.

NO

(at the initiation of the dossier, no test was available)

6c

Are there data from a non-validated suitable in vitro test(s), which provide sound conclusive evidence that the substance is corrosive/ irritant?

NO

 

Weight-of- Evidence analysis

7

The “elements” described above may be arranged as appropriate. Taking all available existing and
relevant data mentioned above (Elements 1-6) into account, is there sufficient information to make a decision on whether classification/labelling is necessary, and –if so –how to classify and label?

NO

 

New in vitro test for corrosivity

8

Does the substance demonstrate corrosive properties in (an) EU/OECD adopted in vitro test(s) for skin corrosion?

NO

 

New in vitro test for irritation

9

Does the substance demonstrate irritating or non-irritating properties in (an) EU/OECD adopted in vitro test(s) for skin irritation?

YES

 => an Episkin test for irritation was initiated (Bottom-up strategy - substance expected to be non corrosive). The conclusion of this Episkin test is sufficient to conclude on C&L (viability = 101,8 % <=> Not a skin irritant)

New in vivo test for corrosion/irritation

10

To be used only as a last resort

NO

In vivo testing should not be conducted in this case since the substance falls under the scope of the specific in vitro tests performed, and there are no substance-specific limitations on use of those tests. An adaptation according to Annex XI to the REACH Regulation is included in this dossier.

The in vitro skin irritation study (Envigo, 2016, Rel.1) was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKIN reconstructed human epidermis model. The quality criteria required for acceptance of results in the test were satisfied. The relative mean viability of the test item treated tissues was 101.8 %, after the 15‑minute exposure period. With a tissue viability > 50%, the test material was considered to be non-irritant to skin.

Eye irritation:

Since no key study was identified on the registered substance, the testing and assessment strategy, as described in ECHA R.7a Endpoint specific guidance (December 2016), was used to evaluate the eye damage/irritation potential of the registered substance:

 

Element

Information

Conclusion

Comments

Conclusion of the information strategy on skin corrosion/irritation

0

Is the substance classified as a skin corrosive?

NO

 

Existing data on physico
- chemical properties

1a

Is the substance spontaneously flammable in contact with air (pyrophoric) or water at room temperature?

NO

 

1b

Is the substance an organic hydroperoxide or an organic peroxide?

NO

 

1c

Is the pH of the substance ≤ 2.0 or ≥ 11.5?

NO

 

1d

Are there other physical or chemical properties that indicate that the substance causes serious eye damage or eye irritation?

NO

 

Existing human data

2

Are there adequate existing human data which provide evidence that the substance has the potential to cause serious eye damage or eye irritation?

NO

 

Existing animal data from corrosion/irritation studies

3

Are there data from existing studies on corrosion and irritation in laboratory animals, which provide sound conclusive evidence that the substance is a corrosive, irritant or non-irritant?

NO

 

Existing/new (Q)SAR data and read-across

4

Are there structurally related substances (suitable “read-across” or grouping), which are classified as causing serious eye damage/eye irritation, or indicating that the substance is non-irritant, or do valid (Q)SAR methods indicate serious eye damage/eye irritation or non-irritation of the substance?

NO

Not applicable - UVCB substance

Existing in vitro data

5a

Has the substance demonstrated serious eye damage, eye irritation or non-irritating properties in an EU/OECD adopted in vitro test?
Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.

NO

 

5b

Are there acceptable data from (a) non-validated suitable in vitro test(s), which provide sound evidence that the substance causes serious eye damage/eye irritation?

NO

(at the initiation of the dossier, no test was available)

Weight-of- Evidence analysis

6

The “elements” described above may be arranged as appropriate. Taking all available existing and relevant data mentioned above (Elements 0 – 5) into account, is there sufficient information to make a decision on whether classification/labelling is necessary, and – if so – how to classify and label?

NO

 

New in vitro tests for serious eye damage/eye irritation (Annex VII to the REACH Regulation)

7a

Does the substance demonstrate serious eye damage, eye irritation or non-irritant properties in (an) EU/OECD adopted in vitro test(s) for the eye hazard charaterisation?
Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions of Annex XI are met.

NO

 

8b

Does the substance demonstrate serious eye damage or eye irritant properties in (a) non-validated suitable in vitro test(s) for serious eye damage/eye irritation?

NO

 => an ICE assay was initiated: no prediction can be made
=> an EpiOcular assay was initiated: potentially requiring classification and labelling (mean percent viability < 60%)

New in vivo test for serious eye damage/eye irritation as a last resort (Annex VIII to the REACH Regulation)

8b

Does the substance demonstrate serious eye damage or eye irritation in an OECD adopted in vivo test?

NO

In vivo testing should not be conducted in this case since the substance is submitted at Annex VII level to the REACH regulation

An ICE assay (Envigo, 2016, Rel.1) was conducted according to the OECD guideline No. 438 and in compliance with GLP. The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 1.3, corresponding to the ICE class II;

- mean score of fluorescein retention: 1.0, corresponding to the ICE class II;

- maximal mean corneal swelling: 20.79% corresponding to the ICE class III.

Therefore no prediction can be made according to the combination of the three endpoints. As a consequence, further in vitro testing was conducted following the top-down approach (Scott et al., 2010) to conclude on eye irritation classification.

The EpiOcular test (Envigo, 2017, Rel.1) was conducted according to the OECD guideline No. 492 and in compliance with GLP. The quality criteria required for acceptance of results in the test were satisfied. The tissue viability of the test item was 32.2% after the 6 -hour exposure period. With a percentage of tissue viability < 60%, the test item require classification for eye irritation or eye damage.

Following the top-down approach, taking into account the ICE assay (not eye damage), the EpiOcular assay (not non-classified) and the absence of skin irritation; the substance is considered to be an eye irritant (Scott et al. 2010).

Reference:

Scott L, Eskes C, Hoffmann S, Adriaens E, Alepée N, Bufo M, Clothier R, Facchini D, Faller C, Guest R, Harbell J, Hartung T, Kamp H, Varlet BL, Meloni M, McNamee P, Osborne R, Pape W, Pfannenbecker U, Prinsen M, Seaman C, Spielmann H, Stokes W, Trouba K, Berghe CV, Goethem FV, Vassallo M, Vinardell P and Zuang V (2010) A proposed eye irritation testing strategy to reduce and replace in vivo studies using Bottom-Up and Top-Down approaches. Toxicol In Vitro 24:1-9.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available information, the substance should be classified as eye irritant Category 2 (H319: Causes serious eye irritation) according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).

No additional self-classification is proposed regarding skin irritation according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).

No data was available regarding respiratory irritation.