Reaction mass of (2Z)-5-[(1R,3R,6S)-2,3-dimethyltricyclo[2.2.1.02,6]hept-3-yl]-2-methyl-2-penten-1-ol and (2Z)-2-methyl-5-[(1S,2R,4R)-2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl]-2-penten-1-ol

Endpoint summary

Description of key information

Skin irritation: not corrosive (EPIDERM, OECD 431, GLP, K, rel.1) but irritating (EPISKIN, OECD 439, GLP, K, rel. 1)

Eye irritation: not irritating (ICE, OECD 492, GLP, K, rel.1)

Respiratory irritation: No data was available.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11-13 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 431 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

18 June 2019

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Monitoring Programme (inspected on 21/08/2018 / signed on 19/11/2018)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: cells obtained from a single or multiple donors

Details on animal used as source of test system:

Not applicable

Justification for test system used:

Following the REACH top-down strategy, the EPIDERM™ Reconstructed Human Epidermis Model method was used to assess skin corrosion.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit, supplied by MatTek
- Tissue batch number: 30893
- Delivery date: 11 December 2019
- Date of initiation of testing: 12 December 2019

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: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT 4500 microplate reader
- Wavelength: 570 nm
- Filter:Yes
- Filter bandwidth: 10 nm
- Linear OD range of spectrophotometer: upper limit 2.6

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.895 +/- 0.072 (Acceptance criteria: 1.0-3.0) <=> Pass
- Barrier function: 4.85 hrs (Acceptance criteria: 4.77-8.72 hrs) <=> Pass
- Morphology: Appropiate formation of the epidermal barrier, presence of functional stratum corneum, viable basal cell layer, and intermediate spinous and granular layers
- Contamination: Sterile (Acceptance criteria: No contamination) <=> Pass
- Reproducibility: Coefficient of Variation between replicates was < 30%

NUMBER OF REPLICATE TISSUES: 2

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of 8.0 N Potassium Hydroxide was used as supplied

Duration of treatment / exposure:

Tissues were treated with the test item for exposure periods of 3 and 60 minutes.

Duration of post-treatment incubation (if applicable):

3 h with MTT

Number of replicates:

Duplicate tissues for test item, negative and positive controls

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

114.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

4.9

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure

Value:

116.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

4.3

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:
All values for negative and positive controls were within the historical range (historical data for negative and positive controls obtained by the laboratory in the previous six months).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 1.866 for the 3 Minute exposure period and 2.008 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 4.3% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied .

Table 7.3.1/1: Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

 

Tissue	Exposure Period	MeanOD570of individual tissues	Mean OD570of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.908	1.866	0.059	3.2	100*
		1.824
	60 Minutes	1.946	2.008	0.087	4.3
		2.069
Positive Control	3 Minutes	0.102	0.091	0.016	na	4.9
		0.080
	60 Minutes	0.090	0.086	0.006	na	4.3
		0.082
Test Item	3 Minutes	2.254	2.135	0.169	7.9	114.4
		2.015
	60 Minutes	2.201	2.333	0.187	8.0	116.2
		2.465

OD=Optical density

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

na=        Not applicable

Relative mean viability (%) = (Mean OD570 of test item / Mean OD570 of negative control) x 100

Coefficient of Variation = (standard deviation / mean OD of duplicate tissues) x 100

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin, according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

An in vitro skin corrosion study was performed according to the OECD Guideline 431 and in compliance with GLP, using the EPIDERM^TM reconstructed human epidermis model.

 

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT-loading each tissue was placed in 2 mL of isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 570 nm (OD₅₇₀).

 

The MTT solution containing the test item did not turn blue and indicate the test item did not reduce MTT. The solution containing the test item did not become colored and indicate the test item did not have the potential to cause color interference.

 

The relative mean viability of the test item treated tissues was 114.4 and 116.2% after 3 and 60 minutes exposure, respectively. The relative mean viability of the negative control treated tissues was set to 100 % after 3 and 60 minutes exposure. The relative mean viability of the positive control treated tissues was 4.9 and 4.3% after 3 and 60 minutes exposure, respectively.

 

The quality criteria required for acceptance of results in the test were satisfied.

 

The test item was considered to be non-corrosive to the skin, according to the Regulation (EC) No 1272/2008 (CLP) and to the GHS.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 December - 20 January 2020

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

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

18 June 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

06 July 2012

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Monitoring Programme (inspected on 21/08/2018 / signed on 19/11/2018)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: cells obtained from adult donors

Justification for test system used:

Following the REACH top-down strategy, as the EPIDERM test for corrosion was negative, the EPIDERM™ Reconstructed Human Epidermis Model method was used to assess skin irritation.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, Episkin Laboratories, Lyon, France
- Tissue batch number(s): 20-EKIN-003
- Delivery date: 14 January 2020
- Date of initiation of testing: 15 January 2020
- Date of scoring: 20 January 2020

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: 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. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each wellsidual test or control items and then incubated in 2mL maintenance medium for 42 h at 37 °C, 5 % CO2 in air. Number of washing steps not reported.
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT 4500 microplate reader
- Wavelength: 570 nm
- Filter: without reference filter
- Linear OD range of spectrophotometer: upper limit of accuracy determined to be at an OD of 2.6.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: IC50 = 2.3 mg/ml ( ≥ 1.5 mg/ml)
- Morphology: Multi-layered, highly differenciated epidermis consisting of aorganized basal, spinous and granular layers and a multilayered stratum corneum. Number of cell layers: 7 (≥ 4).
- Contamination:
On cells from of the donor: absence of bacteria, fungus and mycoplasma
On blood of the donor: absence of HIV1 & 2 & hepatitis C antibodies; absence of hepatitis B antigen HBs
- Reproducibility: All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory obtained in the previous six months. This was taken to show the correct functioning of the test system.

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if relative mean tissue viability is ≤ 50% after 15 minutes of exposure.
- The test substance is considered to be non-irritating to skin if relative mean tissue viability is > 50% after 15 minutes of exposure.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- The test item was used as supplied (undiluted).
- Amount(s) applied (volume or weight with unit): 10 µL

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

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

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Triplicate tissues for test substance, negative and positive controls

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minute exposure period and 42 h post-exposure incubation period

Value:

49

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

6%

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:
In order to confirm the integrity of the test system a comparison was made with historical data for negative and positive controls obtained by the laboratory in the previous six months.
All values for negative and positive controls were within the historical range and this was taken to indicate the adequate functioning of the test system.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 0.851 and the standard deviation value of the viability was 4.5%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 6.0% relative to the negative control treated tissues and the standard deviation value of the viability was 0.6%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 6.1%. The test item acceptance criterion was therefore satisfied .

Table 7.3.1/1: EpiSkin™ results

Item	OD570of tissues	Mean OD570of triplicate tissues	±SDof OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.894	0.851	0.038	105.1	100*	4.5
	0.822			96.6
	0.838			98.5
Positive Control Item	0.046	0.051	0.005	5.4	6.0	0.6
	0.050			5.9
	0.056			6.6
Test Item	0.459	0.417	0.052	53.9	49.0	6.1
	0.432			50.8
	0.359			42.2

OD=Optical Densit

SD=       Standard deviatio

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

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the experimental conditions of this study, the test substance is classified as skin irritant Category 2 according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

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

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 hours. 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 570 nm.

 

The relative mean viability of the test item treated tissues was49.0% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period.

The criteria required for acceptance of results in the test were satisfied.

 

Under the experimental conditions of this study, the test substance is classified as skin irritant Category 2 according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.

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:

15 January 2020

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

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)

Version / remarks:

25 June 2018

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Monitoring Programme (inspected on 21/08/2018 / signed on 19/11/2018)

Species:

chicken

Strain:

other: Spring chickens (Gallus Gallus e.g. Ross 308 Broiler)

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Spring chickens (Gallus Gallus e.g. Ross 308 Broiler), were supplied by Baileys Turkeys Ltd., Cheshire, UK
- Number of animals: 8
- Characteristics of donor animals (e.g. age, sex, weight): The chickens weighed approximately 3 kg and were approximately 56 days old prior to being humanely killed for human consumption.
- 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.
- Time interval prior to initiating testing: The time interval between collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was minimized although all eyes had to fall within the acceptance criteria identified in the test guideline.
Date / Time Of Slaughter: 15.01.2020 0710 am
Date / Enucleation End Time: 15.01.2020 0940 am
- indication of any existing defects or lesions in ocular tissue samples: none
- Indication of any antibiotics used: none

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): 0.03 mL of test item (undiluted)

Duration of treatment / exposure:

The test item was applied for 10 seconds and then rinsed from the eye using 20mL of isotonic saline

Duration of post- treatment incubation (in vitro):

Evaluation of the corneas at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes have been decontaminated

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
Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected.
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 mean 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.

EQUILIBRATION AND BASELINE RECORDINGS
After the approval process the eyes were incubated for approximately 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
The test item and positive control item groups consisted of three eyes and the negative control item consisted of two eyes.

NEGATIVE CONTROL USED
0.03 mL of the negative control item, Sodium chloride 0.9% w/v, was applied to the cornea of each negative control eye.

POSITIVE CONTROL USED
0.03 g of the positive control item, Benzalkonium chloride (5% v/v), was similarly applied to the cornea of each positive control eye.

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.

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.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 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.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Yes
- Damage to epithelium based on fluorescein retention: Yes
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Yes (e.g. pitting, sloughing or roughening of the epithelium).
- Macroscopic morphological damage to the surface: Yes

SCORING SYSTEM:
- Mean corneal swelling (%): Percentage corneal swelling was assessed from corneal thickness measurements. The calculation was expressed in the following formula:
[(corneal thickness at time (t) / corneal thickness at time = 0) / corneal thickness at time =0] x 100
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.
- 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: The mean fluorescein retention scores for all test eyes are calculated at the 30 minute time interval only. These measurements are used for the overall classification for each test item.
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

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

Remarks:

maximal mean score

Run / experiment:

10 seconds exposure

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.8 (ICE Class II)

Positive controls validity:

valid

Remarks:

4.0 (ICE Class IV)

Remarks on result:

no indication of irritation

Remarks:

ICE Class II

Irritation parameter:

fluorescein retention score

Run / experiment:

10 seconds exposure

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.5 (ICE Class I)

Positive controls validity:

valid

Remarks:

3.0 (ICE Class IV)

Remarks on result:

no indication of irritation

Remarks:

ICE Class II

Irritation parameter:

percent corneal swelling

Remarks:

Maximal mean

Run / experiment:

10 seconds exposure

Value:

1.61

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

4.92 (ICE Class I)

Positive controls validity:

valid

Remarks:

44.63 (ICE Class IV)

Remarks on result:

no indication of irritation

Remarks:

ICE Class II

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:
1/ Corneal Opacity Scores
Very faint opacity was noted in one negative control treated eye.
Scattered or diffused areas of opacity were noted in one negative control treated eye.
Complete corneal opacity was noted in all positive control treated eyes.
Very faint opacity was noted in one test item treated eye.
Scattered or diffused areas of opacity were noted in two test item treated eyes.
No morphological effects were noted in the test item, positive or negative control item treated eyes apart from a sunken cornea noted in one test item treated eye.
2/ Fluorescein Retention Scores
Very minor single cell staining was noted in the negative control treated eyes.
Confluent large areas of the cornea retaining fluorescein was noted in all positive control treated eyes.
Very minor single cell staining was noted in one test item treated eye.
Single cell staining scattered throughout the treated area of the cornea was noted in two test item treated eyes.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes
In order to confirm the acceptability of the test, a comparison was made with historical control data for negative and positive controls obtained by the laboratory (Annex 2). The test was considered acceptable as the concurrent negative and positive controls were identified as GHS Non-Classified and GHS Category 1 respectively.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the negative control is identified as GHS-Non Classified
- Acceptance criteria met for positive control: Yes, the positive control is identified as GHS Category 1
- Range of historical values if different from the ones specified in the test guideline: In order to confirm the acceptability of the test, a comparison was made with historical control data for negative and positive controls obtained by the laboratory. The test was considered acceptable as the concurrent negative and positive controls were identified as GHS Non-Classified and GHS Category 1 respectively.

Table 7.3.2/3: Individual Scores and Mean Scores for Corneal Effects – Test Item

End Point		Eye Number	Time (after decontamination)
			0 minutes	30 minutes	75 minutes	120 minutes	180 minutes	240 minutes
Corneal Opacity		3A	0.5	1	1	1	1	1
		6A	0.5	0.5	0.5	0.5	0.5	0.5
		8A	0.5	0.5	1	1	1	1
Mean			0.5	0.7	0.8	0.8	0.8	0.8
ICE Class			II
Fluorescein Retention		3A		1
		6A		0.5
		8A		1
Mean				0.8
ICE Class			II
Corneal Thickness		3A	0.60	0.60	0.60	0.64	0.64	0.64
		6A	0.65	0.62	0.62	0.60	0.60	0.61
		8A	0.61	0.60	0.62	0.63	0.63	0.64
Mean			0.62	0.61	0.61	0.62	0.62	0.63
Mean Corneal Swelling (%)				-2.15	-1.08	0.54	0.54	1.61
ICE Class			I
Epithelium Condition	3A			N	N	N	N	SC
	6A			N	N	N	N	N
	8A			N	N	N	N	N
ICE Classes Combined:			1 x I, 2 x II
Classification:			No Category

N= Normal

SC= Sunken Cornea

N= Normal

TA = Test item adherance

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, the test item is not classified according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

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 corrosivity or severe irritancy potential of the test item as measured by its ability to induce toxicity in an enucleated chicken eye. 

0.03 mL of the test item was applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item. A further two enucleated eyes were treated with negative control item

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

- maximal mean score of corneal opacity: 0.8, corresponding to the ICE class II;

- mean score of fluorescein retention: 0.8, corresponding to the ICE class II;

- maximal mean corneal swelling: 1.61 % at 120 min after treatment corresponding to the ICE class I.

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, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe irritant", as expected.

Under the test conditions, the test item is not classified according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation / corrosion:

Since no key study was identified on the substance, the testing and assessment strategy, as described in ECHA R.7a Endpoint specific guidance (July 2017), was used to evaluate the skin corrosion/irritation potential of the 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?	YES	Predicted to be "Irritating to Skin" according to Danish QSAR Database and Toxtree v3.1.0
	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	(at the initiation of the dossier, no test was available)
	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? 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	=> an Epiderm test for corrosion was initiated Conclusion: Not corrosive to skin
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? 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.	YES	=> an Episkin test for irritation was initiated. Conclusion: Skin irritant
New in vivo test for serious eye damage/eye irritation as a last resort (Annex VIII to the REACH Regulation)	10	Does the substance demonstrate corrosive or irritant properties in an EU/OECD adopted in vivo test?	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.

Multiple Quantitative Structure Activity Relationship (QSAR) models were used to predict the Skin Irritation/Corrosion potential of the registered substance. These QSAR models have been designed to be used for regulatory purposes and based on the QSAR results, this report predicts the consensus endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following the OECD Guideline for Testing of Chemicals No. 404: Acute Dermal Irritation/Corrosion.

This prediction was performed using the following QSAR models:

• Danish QSAR Database

• Toxtree v3.1.0

The final consensus result for skin irritation/corrosion was derived by applying a consensus method on the reliable results derived for individual models.

Based on multiple QSAR models applied, the registered substance was predicted as irritant to the skin.

Nevertheless, in vitro studies were performed to confirm this prediction:

1/ An in vitro skin corrosion study was performed according to the OECD Guideline 431 and in compliance with GLP, using the EPIDERM reconstructed human epidermis model.

The relative mean viability of the test item treated tissues was 114.4 and 116.2% after 3 and 60 minutes exposure, respectively. Under the test conditions, the test item was considered to be non-corrosive to the skin.

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

The relative mean viability of the test item treated tissues was 49.0% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period. Under the experimental conditions of this study, the test substance is a skin irritant.

These studies confirm the QSAR predictions for the substance. Based on the available data, the substance is classified as a skin irritant.

Eye irritation:

Since no key study was identified on the substance, the testing and assessment strategy, as described in ECHA R.7a Endpoint specific guidance (July 2017), was used to evaluate the eye corrosion/irritation potential of the 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	Predicted to be "Irritating to Skin" according to OECD Toolbox v4.3
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?	YES	=> an ICE assay was initiated. Conclusion: not an eye irritant
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 at Annex VII level

A Quantitative Structure Activity Relationship (QSAR) models was initially used to predict the Eye Irritation/Corrosion potential of the substance.

This QSAR model has been designed to be used for regulatory purposes and based on the QSAR results, this report predicts the consensus endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following the OECD Guideline for Testing of Chemicals No. 405: Acute Eye Irritation/Corrosion.

This prediction was performed using the following QSAR model: OECD QSAR Toolbox v4.3 (profiling results)

Based on QSAR model applied, the substance was predicted as irritant to the eyes.

Nevertheless, an in vitro eye irritation study (ICE, OCD 438/GLP, Covance, 2020, rel.1) was performed to confirm this prediction.

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

- maximal mean score of corneal opacity: 0.8, corresponding to the ICE class II;

- mean score of fluorescein retention: 0.8, corresponding to the ICE class II;

- maximal mean corneal swelling: 1.61 % at 120 min after treatment corresponding to the ICE class I.

The substance is classified as "No category".

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.

Based on the recently performed study, the classification has been changed from "EDI2" to "not classify".

Justification for classification or non-classification

Harmonized classification:

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

Self classification:

Based on the available data, additional self-classification is proposed regarding both skin and eye irritation:

- skin irritant Category 2 according to the CLP and to the GHS

- not classified for eye irritation according to the CLP and to the GHS

No data was available regarding respiratory irritation.