Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate

Endpoint summary

Description of key information

Skin irritation: Key study. Method according to the OECD 439 and EU method B.46. GLP study. The mean percent tissue viability of the SkinEthic TM RHE/S17 model (Episkin SA, Lyon) treated tissues was 64.1%. Therefore, the test item should be considered a non-irritant.

Eye irritation: Based on the available data, the test item is not irritating to the eyes.

Weight of Evidence. Test method according to the OECD 438 and EU method B.48. GLP study. According to the overall In vitro classification, no prediction can be made with the ICE test method since the combination of the 3 endpoints for the test item were 1 x IV, 1x II, 1 x I.

Weight of Evidence. Test method according to the OECD 492. GLP study. The mean percent viability of the EpiOcular ^TM tissues treated with the test item 84.34%. Based on the results, the test item is non-irritant to Reconstructed human Cornea-like Epithelium. Therefore, the test item is considered to be non-irritant to the eyes and should not be classified under CLP.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 16th, 2018 to May 31st, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Justification for test system used:

SkinEthic TM RHE/S17 model (Episkin SA, Lyon) has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No.439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic TM RHE/S17 model (Episkin SA, Lyon)
- Tissue batch number(s): 18-RHE-057
- Delivery date: 29 May 2018
- Expiration date: 4 June 2018
- Date of initiation of testing: 16 May 2018

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 x 1 mL of DPBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 μl of a MTT solution at 1.0 mg/mL
- Incubation time: 3 h at 37ºC, 5% CO2
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek)
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: The linearity range of optical density measured is validated for an optical density between 0 and 2.0.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD = 1.2 (CV=3.0%) specification OD > 0.7. Historical negative control mean OD range = 0.653 - 1.194
- Barrier function: 4.7 h (Specification 4 h < ET50 < 10 h)
- Morphology: 5.5 cell layers, absence of significant histological abnormalities, well differentiated epidermis (Specification > 4)
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

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

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 42 minutes of exposure is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes of exposure is greater than 50%.value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage, but the mean disc dye content is greater than or equal to the mean disc dye content of the 10M HCl positive control obtained concurrently.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. if the mean TER value obtained for the test substance is greater than 5 kΩ, or if the mean TER value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage, and the mean disc dye content is well below the mean disc dye content of the 10M HCl positive control obtained concurrently.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 430:

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 μL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL (DPBS –Dutscher - Batch No. 9120318)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

64.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 1

Value:

71.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 2

Value:

72.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 3

Value:

48.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The mean percent viability of the treated tissues was 64.1 %, versus 2.3% in the positive control (5% Sodium Dodecyl sulfate). Therefore, the test item is classified as not irritant. Although the percentage of viability of 1/3 of RHE is ≤ 50% (48.4%) the two others had high viability percentages. (71.7% and 72.3%). Since there is just one epidermis that had borderline results, and according to OECD 439 establishing that if he mean viability gives a clear result, (higher than the range of 50 ± 5%), a second run is not necessary.

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, a full demonstration of proficiency was performed with Episkin-RHE/17 model. As SkinEthic RHE® model is very similar to EpiSkin® model, a reduced validation for the use of SkinEthic RHE ®model was performed (Studies HSMI-2012-A', HSMI-2012-B', HSMI-2012-C' and HSMI-2012-D'). Adequate results were obtained for the evaluated chemicals. Summary of proficiency chemicals tested according to OECD 439 criteria included in the report.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, OD mean of negative control is 0.822 and the acceptability criteria is OD> 0.4x <1.5
- Acceptance criteria met for positive control: Yes, the mean OD for the positive control was 0.19 while the SD was 0.2. These results are valid according to historical data. Historical OD ranged between 0.009-0.0028 and SD between 0.1-0.6.
- Acceptance criteria met for variability between replicate measurements: Yes, the standard deviation of the test item tissue replicates was 13.6%, the acceptability criteria is SD ≤ 18%.

Table 1. Individual and average values of OD after 42 minutes exposure

	Well ID	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD viability	Conclusion
Negative control	SPL 1	0.876	0.871		106.0	100.0	10.1
		0.877
		0.858
	SPL 2	0.695	0.726	0.822	88.4
		0.748
		0.734
	SPL 3	0.850	0.868		105.6
		0.875
		0.878
Positive control	SPL 4	0.020	0.020		2.4	2.3	0.2	Irritant
		0.020
		0.019
	SPL 5	0.020	0.019	0.019	2.3
		0.018
		0.018
	SPL 6	0.016	0.017		2.1
		0.017
		0.017
Test item PH-18/0232	SPL 10	0.602	0.589		71.7	64.1	13.6	Non irritant
		0.589
		0.576
	SPL 11	0.595	0.594	0.527	72.3
		0.595
		0.591
	SPL 12	0.407	0.398		48.4
		0.397
		0.388

# mean of 3 values (triplicate of the same extract)

OD: optival density

The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol.

Acceptability criteria: SD≤18%.

The acceptance criteria were met.

Notes:

- If the viability obtained for the test item is greater than 50%, the test item has to be considered as non irritant.

- If the viability obtained for the test item is less than or equal to 50%, the test item has to be considered as irritant.

Interpretation of results:

GHS criteria not met

Remarks:

EU criteria

Conclusions:

The mean corrected percent viability of the treated tissues was 64.1% . Therefore, the test item has to be considered as non-irritant to the skin.

Executive summary:

The evaluation of the possible irritating effects of the test item has been tested after topical application on in vitro human reconstructed epidermis ( SkinEthic TM RHE/S17 model) in accordance with OECD 439 and EU method B.46, under GLP conditions. The test item was applied, as supplied, at a dose of 16 μL to 3 living reconstructed Human epidermis during 42 minutes, followed by a rinse with 25 mL of DPBS and a 42 h post-incubation period at 37ºC, 5% CO2. Cell viability was measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. Positive and negative control were run in parallel. All acceptability criteria were met. The percent viability of the replicates was 71.7, 72.3 and 48.4%. Although the percentage of viability of 1/3 of RHE is ≤ 50% (48.4%)  the two others had high viability percentages. (71.7% and 72.3%). Since there is just one epidermis that had borderline results, and according to OECD 439 establishing that if the mean viability gives a clear result, (higher than the range of 50 ± 5%), a second run is not necessary. The mean corrected percent viability of the treated tissues was 64.1%, versus 2.3% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item cannot be classified or skin irritation/corrosion according to CLP Regulation no. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

July 2nd, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

October 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse (Etablissement Brun, 33820 Etauliers, France), the chickens were killed for human consumption
- Characteristics of donor animals: spring chickens (approximately 7 weeks old, 1.5 - 2.5 kg)
- Storage, temperature and transport conditions of ocular tissue: Heads were removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. Intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline.
- Time interval prior to initiating testing: The head collection was at 8:30 am and arrived at 10:15 am at the laboratory.
- Indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: No

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

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

No treatment incubation is performed.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Dissection of the eyes: the eyelids were carefully excised and the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.
Selection of the eyes: The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 32.1ºC and 32.3ºC. After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure using sodium fluorescein. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of >0.5; (ii) corneal opacity>0.5, or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.

EQUILIBRATION AND BASELINE RECORDINGS
The eyes examined and approved were incubated between 45 and 60 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e. Time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 30 μL Physiological saline - Dutscher Batch No. 3013132 (1 replicate)

POSITIVE CONTROL USED: 30 μL 5% (w/v) Benzalkonium chloride - Sigma Batch No. BCBQ9761V (3 replicates)

APPLICATION DOSE AND EXPOSURE TIME
30 μL of the test item were applied for 10 seconds

OBSERVATION PERIOD
Pretreatment and at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL of physiological saline at ambient temperature
- Indicate any deviation from test procedure in the Guideline: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: It was measured using a HaagStreit BP900 slit-lamp microscope with depth measuring device no. I. It was calculated using the area of the cornea that was most densely opacified for scoring.
- Damage to epithelium based on fluorescein retention: It was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: It was measured using a HaagStreit BP900 slit-lamp microscope with depth-measuring device no. I, the slit-width was set at 91/2, equalling 0.095 mm. The mean percentage of corneal swelling was calculated for all observation time points.
- Macroscopic morphological damage to the surface: include "pitting" of corneal epithelial cells, "loosening" of epithelium, "roughening" of the corneal surface and "sticking" of the test item to the cornea. The classification of these findings is subjective according to the interpretation of the investigator.

SCORING SYSTEM:
- Mean corneal swelling (%): (corneal thickness at time t -corneal thickness at time=0/corneal thickness at time=0)x100.
- Mean maximum opacity score: 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.

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. All endpoints were evaluated separately to generate an ICE class for each endpoint and then combined to generate an Irritancy Classification for the test item. The ICE classes were assigned based on a predetermined range.
ICE classification criteria for corneal thickness: 0 to 5 = class I; >5 to 12= class II; >12 to 18 (>75 min after treatment)=class II; >12 to 18 (≤75 min after treatment)=class III;>18 to 26= class III; >26 to 32 (>75 min after treatment)= class III; >26 to 32 (≤75 min after treatment)= class IV; >32=class IV. ICE classification criteria for opacy: 0.0-0.5 = class I; 0.6-1.5= class II; 1.6-2.5 =class III; 2.6-4.0=class IV.
ICE classification criteria for mean fluorescein retention: 0.0-0.5 = class I; 0.6-1.5= class II; 1.6-2.5 =class III; 2.6-3.0=class IV.
Overall in vitro classifications:
No category= 3 x I / 2 x I, 1 x II
No prediction can be made= Other combinations
Category 1 (Corrosive/Severe Irritant)= 3 x IV / 2 x IV, 1 x III / 2 x IV, 1 x II / 2 x IV, 1 x I / Corneal opacity ≥ 3 at 30 min (in at least 2 eyes) / Corneal opacity

Irritation parameter:

cornea opacity score

Run / experiment:

Maximal Mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE Class I

Irritation parameter:

fluorescein leakage

Run / experiment:

Mean

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE class IV

Irritation parameter:

percent corneal swelling

Run / experiment:

Maximal mean

Value:

10

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE class II

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The test is considered accpetable if the negative control is identified as GHS Non-Classified, The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.
- Acceptance criteria met for positive control: The test is considered accpetable if the positive control is identified as GHS Category 1. 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.
- Range of historical values if different from the ones specified in the test guideline: Regular positive controls performed during 2017 had a combination of endpoints 3 x IV, and would be classiied as corrosive, severe irritant. The negative control combinations of endpoints ranged from 2 x I, 1 x II to 3 x I and were all classified as GHS no category.

Table 1. Individual and average values for evaluation of corneal lesions after treatment with the negative control (Physiological saline)

Endpoint measured	Eye No.	0	30	Time (min) 75	120	180	240
Corneal opacity	16	0.0	0.0	0.0	0.0	0.0	0.0
ICE class			I
Fluorescein retention	16	0	0	-	-	-	-
ICE class			I	-	-	-	-
Corneal thickness	16	0.57	0.57	0.57	0.57	0.57	0.57
Corneal swelling (%)	16	-	0	0	0	0	0
ICE class			I
Combination of the 3 Endpoints		3 x I
CLASSIFICATION		No category

Note: No morphological effects were noted, whatever the examination time.

Table 2. Individual and average values for evaluation of corneal lesions after treatment with the positive control ( 5% (w/v) Benzalkonium chloride)

Endpoint measured	Eye No.	0	30	Time (min) 75	120	180	240
	1	0	3	3	3	3	3
Corneal opacity	2	0	3	3	3	3	3
	3	0	3	3	3	3	3
Mean		0.0	3.0	3.0	3.0	3.0	3.0
ICE class			IV
	1	0.5	3	-	-	-	-
Fluorescein retention	2	0.5	3	-	-	-	-
	3	0.5	3	-	-	-	-
Mean		0.5	3.0	-	-	-	-
ICE class			IV	-	-	-	-
	1	0.56	0.66	0.71	0.80	0.82	0.84
Corneal thickness	2	0.60	0.71	0.78	0.82	0.82	0.83
	3	0.61	0.72	0.76	0.84	0.85	0.86
	1	-	18	27	43	46	50
Corneal swelling (%)	2	-	18	30	37	37	38
	3	-	18	25	38	39	41
Mean		-	18	27	39	41	43
ICE class			IV
Combination of the 3 Endpoints		3 x IV
CLASSIFICATION		Category 1 : Corrosive / Severe irritant

Blisters on the cornea noted 30 minutes post dose in eyes No.1, 2, 3.

Table 3. Individual and average values for evaluation of corneal lesions after treatment with the test item.

Endpoint measured	Eye No.	0	30	Time (min) 75	120	180	240
	7	0	0	0	0	0	0
Corneal opacity	8	0	0	0	0	0	0
	9	0	0	0	0	0	0
Mean		0.0	0.0	0.0	0.0	0.0	0.0
ICE class			I
	7	0.5	3	-	-	-	-
Fluorescein retention	8	0.5	3	-	-	-	-
	9	0.5	3	-	-	-	-
Mean		0.5	3.0	-	-	-	-
ICE class			IV	-	-	-	-
	7	0.57	0.63	0.64	0.64	0.64	0.66
Corneal thickness	8	0.58	0.62	0.62	0.62	0.62	0.64
	9	0.60	0.63	0.63	0.63	0.63	0.63
	7	-	11	12	12	12	16
Corneal swelling	8	-	7	7	7	7	10
(%)	9	-	5	5	5	5	5
Mean		-	7	8	8	8	10
ICE class			II
Combination of the 3 Endpoints		1 x IV, 1 x II, 1 x I
CLASSIFICATION		No prediction can be made

Note: No morphological effects were noted, whatever the examination time.

Interpretation of results:

study cannot be used for classification

Conclusions:

The combination of the 3 endpoints fot the test item was 1 x IV, 1x II, 1 x I, according to OECD 438. Therefore, no prediction can be made.

Executive summary:

The eye irritation of the test item has been evaluated using the Isolated Chicken Eye test method, according to OECD 438 and EU method B.48, under GLP conditions. 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 twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control ( 5% (w/v) Benzalkonium chloride) and one eye with a negative control (physiological saline). Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention and were evaluated at pretreatment and at 30, 75, 120, 180 and 240 minutes post-dose. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438. Positive control and negative control results were valid. The mean corneal opacity for the test item treated corneas was 0, corresponding to ICE category class I; the mean score of fluorescein retention was 3, corresponding to ICE category class IV; and the mean corneal swelling was 10%, corresponding to ICE class II. The combination of the 3 endpoints for the test item was 1 x IV, 1x II, 1 x I, according to OECD 438. Therefore, no prediction can be made.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From September 11th, 2018 to September 20th, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

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:

October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: EpiOcular (TM).

Remarks:

batch No. 27068

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcular(TM) model and the corresponding test method have been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline (OECD No. 492), and the method is applicable to mixtures, solids, liquids, semi-solids and waxes. Therefore, it was considered to be suitable for this study.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The test was performed using EpiOcular(TM) Reconstructed Human Corneum Epithelium (RhCE) from MatTek Corporation. The system consists of normal, human-derived corneal keratinocytes which have been cultured in an air-liquid interphase to form a multilayered highly differentiated model of the human ocular epithelium. Ultrastructurally, the EpiOcular system closely parallels human cornea, thus providing a useful in vitro means to assess ocular toxicology. The certificate of Analysis of the test system is included in the report. The tissue viability and barrier function tests are within the acceptable ranges and indicate the appropiate formation of the mucosal barrier and a viable basal cell layer.
The cells used to produce EpiOcular (TM) tissue are screened for potential biological contaminants:
- HIV-1 virus - oligonucleotide-directed amplification: Not detected.
- Hepatitis B virus - oligonucleotide-directed amplification: Not detected.
- Hepatitis C virus - oligonucleotide-directed amplification: Not detected.
- Bacteria, yeast and other fungi - long term antibiotic, antimycotic free culture: Not detected.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 μL

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

2 tissues per test substance and for each control were used.

Details on study design:

- Details of the test procedure used: the tissues were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed assay medium (MatTek Corporation, batch No. 091718ALA) and incubated during 19 hours and 45 minutes at standard culture conditions. After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+ Free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes. The test item was applied as supplied, at the dose of 50 μL, to the entire surface of 2 living 0.60 m2 RhCE tissue replicates during 30 minutes at standard culture conditions. After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS (Dutscher - Batch No. 3410618). The rinsed tissues were checked for any colouration and noted to be of comparable colour with the negative control treated tissues (whitish). This rinsing step was followed by a 12 minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue. The RhCE constructs were then incubated for a 2 hours post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2. Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for the appearance of clear cytotoxic effects. The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues. The RhCE constructs were placed in 300 μL of a MTT solution at 1.0 mg/mL for 3 hours at standard culture conditions. The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 18 hours and 73 minutes at 6±3°C in the dark. The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2).
- RhCE tissue construct used, including batch number: EpiOcularTM OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 27068
- Doses of test chemical and control substances used: 50 µL in all cases.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): exposure 30 min; post-exposure immersion 12 min; post-exposure incubation 120 min.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2 tissues per test substance and for each control were used.
- Wavelength and bandpass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): Wavelength: 570 nm, of OD, was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.
- Description of the method used to quantify MTT formazan: The RhCE constructs were placed in 300 μL of an MTT solution at 1.0 mg/mL for 3 hours at standard culture conditions. The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 18 hours and 73 minutes at 6±3°C in the dark. The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2).
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
The OD values obtained with the replicate tissue extracts for each test item were used to calculate the mean percent tissue viability normalized to the negative control, which was set to 100%. The percentage of tissue viability cut-off value distinguishing classified from non-classified test items is 60%. Results are interpreted as follows: The test item is identified as not requiring classification and labelling according to UN GHS No Category: if the mean percent tissue viability after exposure and post-exposure incubation is > 60%. In this case, no further testing in other test methods is required.
The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1): if the mean percent tissue viability after exposure and post-exposure incubation is ≤ 60%.
When the final mean percent tissue viability is ≤ 60%, further testing with other test methods will be required because the RhCE test method shows a certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Historical negative control mean OD range =0.585-1.563, historical positive control mean OD range =0.025-0.693
- Complete supporting information for the specific RhCE tissue construct used: Keratinocyte Strain 4F1188, Tissue Viability 2.447±0.065b (Validity criteria 1.1-3.0), Barrier function 21.1 min (Validity criteria: 12.2-37.5 min), sterility= no contamination. Manufacture date: September 18th, 2018, Received: September 18th, 2018.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: yes, 12 out of 15 substances were tested and presented adequate results.
- Positive and negative control means and acceptance ranges based on historical data: Negative control mean viability=100%, Postive control=11.75. Negative control, OD values of the two replicates should be in the range > 0.8 and < 2.5. As the extract was diluted at 50% just before the OD measurement, the acceptability criteria should be in the range > 0.4 and < 1.25 for the negative control. Tissues treated with the positive control substance should show mean tissue viability < 50%.
- Acceptable variability between tissue replicates for the test chemical: Difference viability should be less than 20%.

Irritation parameter:

other: Cell viability

Run / experiment:

Mean

Value:

84.34

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Variability: 0.17%

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes (attached).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (mean cell viability = 100%; SD = 7.46%)
- Acceptance criteria met for positive control: yes (mean cell viability = 11.75 %; SD = 0.51%)
- Range of historical values if different from the ones specified in the test guideline: no.

Table 1. Summary of results.

	Well ID	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Difference of viability %	Conclusion
Negative control	SPL 1	1.045 1.160 1.162	1.122	1.166	96.27	100.00	7.46
	SPL2	1.199 1.221 1.208	1.209		103.73
Positive control	SPL 3	0.159 0.134 0.129	0.140	0.137	12.01	11.75	0.51	UN GHS Category 2 or 1
	SPL 4	0.139 0.133 0.131	0.134		11.50
Test item PH-18/0232	SPL 5	0.970 0.997 0.987	0.984	0.983	84.43	84.34	0.17	No Category
	SPL 6	0.960 1.001 0.985	0.982		84.26

Interpretation of results:

GHS criteria not met

Remarks:

UN GHS

Conclusions:

The mean corrected percent viability of the treated tissues was 84.34%. Therefore, does not require classification for eye irritation or serious eye damage.

Executive summary:

An in vitro study according to OECD 492, under GLP conditions was performed to assess the eye irritation potential of the test item. The tissues were incubated at standard culture conditions during 30 minutes. The test item was applied as supplied, at the dose of 50 μL, to the entire surface of 2 living RhCE tissue replicates during 30 minutes at standard culture conditions. After the treatment the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS. Then, a 12 minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue was performed. The RhCE constructs were then incubated for a 2 hours post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37 °C, 5% CO2. The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt. The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 18 hours and 73 minutes at 6 ± 3 ºC in the dark.The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2). Based on the results, mean percent tissue viability after exposure and postexposure incubation was 84.34% versus 11.75% in the positive control (Methyl acetate). Therefore, the test item does not require classification for eye irritation or serious eye damage according to CLP Regulation no. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation: Key study. An In vitro skin irritation test of the test item was performed in a reconstructed human SkinEthic TM RHE/S17 model (Episkin SA, Lyon) , according to OECD 439 and EU method B.46, under GLP conditions. Three epidermis units were treated with 16 mg test item for 42 minutes at room temperature. After 42h postincubation, the viability of each tissue was assessed by incubating the solution with MTT, extracting the precipitated formazan crystals, and determining the OD spectrophotometrically. Under test conditions, the mean per cent viability of the treated tissues was 64.1%. Therefore, the test item has to be considered as non-irritant to the skin.

Eye irritation:

Weight of evidence. An in vitro study was conducted in order to determine the potential severe eye-damaging effects of the test item according to the OECD guideline 438 and EU method B.48 under GLP conditions. Eyeballs isolated from chickens killed for human consumption and were exposed to either 30 μL of the test item, 30 μLo f Benzalkonium chloride (positive control) or 30 μL of physiological saline (negative control). Three eyeballs were used in test item and positive groups, and one for the negative control group. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438. Under experimental conditions, the combination of the 3 endpoints for the test item was 1 x IV, 1x II, 1 x I, according to OECD 438. Therefore, no prediction can be made.

Weight of evidence. An in vitro study according to OECD 492, under GLP conditions, was performed to assess the eye irritation potential of the test item. The tissues were incubated at standard culture conditions for 30 minutes. The test item was applied as supplied, at the dose of 50 μL, to the entire surface of 2 living RhCE tissue replicates during 30 minutes at standard culture conditions. AThe RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt. The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 18 hours and 73 minutes at 6 ± 3ºC in the dark. The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2). Based on the results (mean per cent tissue viability after exposure and postexposure incubation was 84.34% versus 11.75% in the positive control (Methyl acetate). Therefore, does not require classification for eye irritation or serious eye damage. The test item does not require classification for eye irritation or serious eye damage according to CLP Regulation no. 1272/2008.

Justification for classification or non-classification

Skin irritation/corrosion: Based on the available data (mean per cent viability 64.1% in OECD 439), the substance is not classified for skin irritation/corrosion according to CLP Regulation no. 1272/2008.

Eye irritation: Since no prediction could be made based on OECD 438, and based on the available data (mean per cent tissue viability after exposure and postexposure incubation was 84.34% ) provided by OECD 492, the test item does not require classification for eye irritation or serious eye damage according to CLP Regulation no. 1272/2008.