Reaction mass of 1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol and 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)butan-2-one and 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-one

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 September 2017 - 25 September 2017

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:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Amended by EC No. 640/2012 OJ No. L193, 20 July 2012.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd. 03 November 2015

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
- EPISKIN Small Model (TM) (EPISKIN-SM (TM), 0.38 cm^2, Batch no.: 17-EKIN-038); a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded in 12-well plates on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen and cultured for 13 days.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Histology scoring: 21.0 ± 1.2 (CV=5.6%)
- IC50: 2.2 mg/mL
- Expiration date: 25 September 2017

SKIN DISC PREPARATION
- Procedure used: On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 23 hours at 37°C

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37.0 ± 1.0°C (actual: 36.3-46.4°C)
- Humidity: 80 - 100% (actual: 34-94%)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1, with phosphate buffered saline
- Observable damage in the tissue due to washing: no

- Interference with the MTT endpoint was tested before the study by adding 10 μL of the test item to 90 μL of MTT solution (test for color interference) and by adding 25 μL of the test item to 2 mL MTT solution (0.3 mg/mL in PBS).

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT (0.3 mg/mL in PBS)
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570 nm

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test item.

ACCEPTABILITY CRITERIA:
a)  The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST ITEM:
- Amount applied: 25 µL

NEGATIVE CONTOL:
- Amount applied: 25 µL Phosphate buffered saline

POSITIVE CONTROL
- Amount applied: 25 µl
- Concentration: 5% (aq) Sodium dodecyl sulphate
- Re-spread after 7 minutes contact time

Duration of treatment / exposure:

15 ±0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours and 3 hours with MTT

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean of 3 replicates

Value:

19

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean tissue viability: 5.4%

Other effects / acceptance of results:

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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the three tissues of the negative control were within the laboratory historical control data range (i.e. 1.201 ± 0.180) and the SD of the % viability was <18% (i.e. 7.2%)
- Acceptance criteria met for positive control: yes, the mean relative tissue viability of the positive control was <50% (i.e. 5.4%) and the SD of the % viability was <18% (i.e. 1.4%).
- Acceptance criteria met for variability between replicate measurements: yes, the SD calculated from individual % tissue viabilities of the three identically treated replicates was <18% (i.e. 15%).

- Since the mean relative tissue viability for the test item was below 50% the test item is considered to be irritant.

Table 2 Individual OD measurements (570 nm)

	A (OD570)	B (OD570)	C (OD570)
Negative control OD570measurement 1 OD570measurement 2	1.3365 1.3054	1.1564 1.1426	1.2816 1.2394
POLYAMBROL OD570measurement 1 OD570measurement 2	0.1277 0.1251	0.4796 0.4644	0.2158 0.2052
Positive control OD570measurement 1 OD570measurement 2	0.1289 0.1202	0.1018 0.1044	0.0947 0.0907

OD = Optical density

Triplicate exposures are indicated by A, B and C.

Table 3 Historical data for skin irritation studies

	Negative control (absorption; OD570)	Positive control (absorption; OD570)
Range	0.676 – 1.336	0.036 – 0.549
Mean	1.01	0.16
SD	0.16	0.10
n	155	154

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of November 2013 to November 2016.

Interpretation of results:

study cannot be used for classification

Remarks:

Additional information on corrosion is needed for classification.

Conclusions:

In an in vitro skin irritation study, performed according to OECD guideline 439 and GLP principles, Polyambrol was found to be irritant to the skin (mean tissue viability of 19%).

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 January 2018 - 02 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

d.d. 22 January 2018

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin corrosion test system is the EpiDerm Skin Model, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Lot no.: 27912
- Surface: 0.6 cm^2
- Date of analysis: 31 January 2018

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.936 ±0.078 (within acceptance criteria)
- Barrier function: 8.05 hours (within acceptance criteria)
- Contamination: no contamination was detected

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during 3-minute treatment: room temperature
- Temperature used during 1-hour treatment: 36.4 - 37.3°C
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: once with phosphate buffered saline
- Observable damage in the tissue due to washing: no

The test item was checked for possible interference with the MTT endpoint in a previous study. It was shown that the test item did not interfere with the MTT endpoint.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Amount of MTT used: 300 μL
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2 for the test item, the negative control and the positive control for each exposure period

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: one experiment with a 3-minute exposure period and one experiment with a 1-hour exposure period.

DECISION CRITERIA (see table 1 in 'any other information on materials and methods')
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

ACCEPTABILITY CRITERIA
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 μL

NEGATIVE CONTROL
- Amount applied: 50 μL

POSITIVE CONTROL
- Amount applied: 50 μL

Duration of treatment / exposure:

3-minute and 1-hour

Duration of post-treatment incubation (if applicable):

3 hours with MTT

Number of replicates:

2 for each exposure period (4 in total)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

117

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Tissue viability: 15%

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour exposure

Value:

116

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Tissue viability: 8.4%

Other effects / acceptance of results:

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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits (i.e. 1.705 for the 3-minute exposure and 1.782 for the 1-hour exposure)
- Acceptance criteria met for positive control: yes, the mean relative tissue viability following the 1-hour exposure to the positive control was 8.4%.
- Acceptance criteria met for variability between replicate measurements: yes, In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 30% (i.e. ≤18%) indicating that the test system functioned properly

Table 2 Individual OD Measurements at 570 nm

	3-minute application (OD570)        A              B		1-hour application (OD570)        A              B
Negative control OD570measurement 1 OD570measurement 2 OD570measurement 3
	1.8820	1.6157	2.0493	1.6588
	1.8490	1.6478	1.9806	1.6469
	1.8437	1.6423	1.9693	1.6367
Test item OD570measurement 1 OD570measurement 2 OD570measurement 3
	2.0909	1.9658	2.2000	2.0465
	2.0825	2.0105	2.1867	2.0338
	2.0711	1.9976	2.1527	2.0514
Positive control OD570measurement 1 OD570measurement 2 OD570measurement 3
	0.2416	0.3502	0.2051	0.1850
	0.2387	0.3552	0.1984	0.1827
	0.2383	0.3572	0.1992	0.1827

OD = Optical density

Duplicate exposures are indicated by A and B.

Table 3 Historical Control Data for in vitro Skin Corrosion Studies

	Negative control		Positive control
	3-minute treatment (OD570)	1-hour treatment (OD570)	3-minute treatment (OD570)	1-hour treatment (OD570)
Range	1.258 – 2.615	1.371 – 2.371	0.0172 – 0.56	0.046 – 0.339
Mean	1.80	1.82	0.19	0.14
SD	0.26	0.22	0.09	0.05
n	111	110	106	103

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of November 2014 to November 2017.

Interpretation of results:

GHS criteria not met

Remarks:

Not classified according to Regulation (EC) 1272/2008.

Conclusions:

Based on the results of an in vitro skin corrosion study, performed according to OECD guideline 431 and GLP principles, Polyambrol is considered not corrosive (mean tissue viability of 117% and 116% after 3 minutes and 1 hour exposure, respectively) and is therefore not classified according to GHS and Regulation (EC) 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

key study

Study period:

05 September 2017 - 03 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: young cattle, obtained from the slaughterhouse Vitelco, 's-Hertogenbosch, The Netherlands.
- Storage, temperature and transport conditions of ocular tissue: eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Subsequently, eyes were collected an transported in physiological saline in a suitable container under cooled conditions.
- The eyes were checked for unacceptable defects and those exhibiting defects were discarded.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL

NEGATIVE CONTROL: Physiological saline
- Amount applied: 750 µL

POSITIVE CONTROL: Ethanol
- Amount applied: 750 µL

Duration of treatment / exposure:

10 ± 1 minutes (both experiments)

Duration of post- treatment incubation (in vitro):

120 ±10 minutes in cMEM for opacity measurements and subsequently 90 ±5 minutes in sodium-fluorescein for permeability determinations

Number of animals or in vitro replicates:

3 for the negative control, the positive control and the treatment group each for both experiments.

Details on study design:

The experiment was repeated based on a calculation error data. The performed repeat experiment was considered not necessary afterwards. However, the performance of an additional experiment has no impact on the study.

TREATMENT METHOD: The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control or the test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32.0 ± 1°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM.
- POST-EXPOSURE INCUBATION: 120 ±10 minutes in cMEM for opacity measurements and subsequently 90 ±5 minutes in sodium-fluorescein for permeability determinations

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity meter (OP-KIT)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (TECAN Infinite® M200 Pro Plate Reader, OD490). OD490 values of less than 1.500 were used in the permeability calculation.
- Other: possible pH effects of the test substance on the corneas were recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA (see table 1):
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces IVIS >55 is defined as a corrosive or severe irritant (UN GHS: category 1);
For a test substance that induces an IVIS >3 and ≤55, no prediction on irritant potency can be made.

Irritation parameter:

in vitro irritation score

Run / experiment:

First experiment; mean of 3 replicates

Value:

11

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean IVIS: 55

Irritation parameter:

in vitro irritation score

Run / experiment:

Second experiment; mean of 3 replicates

Value:

5.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean IVIS: 57

Other effects / acceptance of results:

- 5 out of the 6 individual corneas resulted in an IVIS > 3 ≤ 55
- The corneas treated with the test item showed opacity values between 3.6 and 9.0 in the first experiment and between 0.4 and 2.6 in the second experiment.
- Permeability values were ranging from 0.108 to 0.726 in the first experiment and ranging from 0.070 to 0.670 in the second experiment.
- IVIS scores were 5.2, 9.3 and 20 (n=3) in the first experiment and 3.6, 10 and 2.2 (n=3) in the second experiment.

OTHER EFFECTS:
- No pH effect of the test item was observed on the rinsing medium in both experiments.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, results were within historical range (IVIS ranging from 1.0 to 1.6 in the first experiment and ranging from 0.2 to 0.7 in the second experiment).
- Acceptance criteria met for positive control: yes, results were within historical range (IVIS ranging from 34 to 73 in the first experiment and ranging from 55 to 59 in the second experiment). Corneas were turbid after 10 minutes of treatment in both experiments.

Table 2 Summary of opacity, permeability and in vitro scores in the first experiment

Treatment	Mean opacity^*	Mean permeability^*	Mean IVIS^*#
Negative control	1.2	0.013	1.4
Positive control	18	2.463	55
Test item	6.3	0.326	11

^*Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.
^#In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

 

Table 3 Summary of opacity, permeability and in vitro scores in the repeated experiment

Treatment	Mean opacity^*	Mean permeability^*	Mean IVIS^*#
Negative control	0.4	0.002	0.4
Positive control	22	2.299	57
Test item	1.2	0.282	5.4

^*Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.
^#In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Table 4 Historical Control Data for the BCOP Studies

	Negative control			Positive control
	Opacity	Permeability	In vitro Irritancy Score	In vitro Irritancy Score
Range	-2.9 – 3.0	-0.016 – 0.042	-2.8 – 3.0	34.7 - 78.2
Mean	0.08	0.01	0.17	56.01
SD	1.04	0.01	1.14	12.51
n	84	77	78	55

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of Aug 2014 to Aug 2017.

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline 437 and GLP principles, no prediction on the classification of Polyambrol can be made (IVIS > 3 ≤ 55).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 December 2017- 22 December 2017

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:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

d.d. 22 January 2018

Species:

human

Details on test animals or tissues and environmental conditions:

TEST SYSTEM:
The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.
- Justification: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 μL
- The test material was directly applied to the tissues which were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS.

POSITIVE CONTROL
- Amount applied: 50 μL Methyl Acetate

NEGATIVE CONTROL
- Amount applied: 50 μL Milli-Q water

Duration of treatment / exposure:

30 minutes ± 2 minutes

Duration of post- treatment incubation (in vitro):

Post-soak: 12 ± 2 minutes; incubation after the post-soak: 120 minutes ± 15 minutes

Number of animals or in vitro replicates:

2 tissues per test item and 2 tissues for the negative and the positive control each.

Details on study design:

TEST PROCEDURE
- RhCE tissue construct used, including batch number: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27410)
- Duration and temperature: before the assay was started the tissues were incubated at standard culture conditions for 30 ± 2 minutes. The tissues were exposed to the test item for 30 minutes ± 2 minutes at 37.0 ± 1.0°C (actual temperature: 36.5-37.3°C). After the exposure period, the tissues were thoroughly rinsed with Ca2+Mg2+-free DPBS (brought to room temperature) to remove residual test item.The post-soak immersion was 12 ± 2 minutes at room temperature in assay medium followed by an incubation of 120 minutes ± 15 minutes at 37°C in assay medium. After incubation the cell cultures were exposed to MTT for 180 ± 10 minutes at 37°C.
- The test item was checked for possible color interference by a 1-hour incubation with sterile Milli-Q water and a 2-3 hour incubation with isopropanol, before the study started. Possible direct MTT reduction was checked by a 3-hour incubation with MTT before the study was started.

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature: 37.0 ± 1.0°C (actual: 36.5 - 37.3°C)
- Humidity: 80-100% (actual: 49-89%), containing 5.0 ± 0.5% CO2

TISSUE VIABILITY MEASUREMENTS
After the exposure and post-soak periods the tissues were incubated with 0.3 mL MTT-medium (1.0 mg/ml) for 180 ± 10 minutes at 37°C. After incubation with MTT-medium the tissues were transferred to a 24-well plate containing 2 mL isopropanol. Formazan was extracted with 2 mL isopropanol refrigerated for 18 ± 2 hours in the dark. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

EVALUATION CRITERIA
- The test chemical 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 more than (>) 60%. In this case no further testing in other test methods is required.
- The test chemical 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 less than or equal (≤) to 60%.

ACCEPTABILITY CRITERIA
- The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.
- The mean relative tissue viability of the positive control should be <50% relative to the negative control.
- The difference between the % tissue viabilities of the two identically treated replicates should be <20%.

Irritation parameter:

other: mean tissue viability (%)

Value:

53

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

mean tissue viability: 28%

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no
- Interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: see 'any other information on results' for historical data.
Results for the positive control were within the historical data range and therefore showing that the test system was functioning properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the two tissues of the negative control was between >0.8 and <2.5 (i.e., 1.52)
- Acceptance criteria met for positive control: yes, the mean relative tissue viability of the positive control was <50% (i.e., <29%).
- The difference between the % tissue viabilities of the two identically treated replicates was <20% (i.e., 4.26, 11.96 and 4.45 for the negative control, the treatment group and the positive control respectively)

Table 1 Historical data for EpiOcular studies

	Negative control (absorption; OD570)	Positive control (absorption; OD570)	Positive control (viability; %)
Range	1.077 – 1.805	0.029 – 0.793	2.11 – 48.25
Mean	1.52	0.42	26.86
SD	0.21	0.23	14.11
n	16	16	16

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of January 2013 to May 2017.

Table 2 Individual OD Measurements at 570 Nm

	A (OD570)	B (OD570)
Negative control OD570 measurement 1 OD570 measurement 2	1.4748 1.4799	1.5420 1.5375
POLYAMBROL OD570 measurement 1 OD570 measurement 2	0.9140 0.8999	0.7461 0.7171
Positive control OD570 measurement 1 OD570 measurement 2	0.4916 0.4912	0.4294 0.4229

OD = Optical density

Duplicate exposures are indicated by A and B.

Interpretation of results:

other: Category 1 or 2

Conclusions:

Based on the results of an in vitro skin corrosion EpiOcular test, Polyambrol is potentially irritant or corrosive to the eyes with a mean cell viability of 53% compared to the negative control. The test item is therefore classified as category 1 or 2 according to GHS and according to Regulation (EC) 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin corrosion/ irritation

in vitro irritation (OECD 439) 15 -min contact: 19% vianility

According to this result, it is not possible to cnclude whatever the substance is corrosive

or iritant (CLP cat. 1 or 2), therefore a second assay was conducted to assess the corrosivity of the substance.

second in vitro assay (OECD 431):

3-min cntact : viability 117%

1hr-contact: 116% viability

Therefore it is concluded that the material has not potential for corrosion on skin but is a skin irritant.

Eye corrosion/ irritation

The BCOP assay gave an IVIS score of 5.4 for the substance, which leads to "inconclusvise" for GHS/ CLP classification.

Therefore, a second assay was conducted.

In an EpiOcular assay (OECD 492), the viability was of 53% (30 -min contact).

In accordance, this assay leads to an "inconclusive" classification.

However, as the BCOP assay tends to over-predict irritation potential for mild or non-irritants (ICCVAM, 2009), and as the score is just above the cut-off value of 3 for non classification, the material is most likely non irritant and not corrosive.

This is supported by the second inconclusive eye irritation test where the viability is just below the cut-off value of 60% for the classification as "no category".