2-ethylhexanoic acid, cerium salt

Endpoint summary

Description of key information

skin irritation: not irritating (OECD 439; GLP)

eye irritation: not irritating (OECD 437; GLP)

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:

2018-01-24 to 2018-01-26

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:

2015-07-28

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)

Version / remarks:

2014-11-07

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-06-05

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in a sealed container

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human epidermal keratinocytes

Cell source:

other: humans

Source strain:

other: not applicable

Details on animal used as source of test system:

not applicable

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human-derived epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely resembles the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

other: Dulbecco's phosphate buffered saline

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-200-SIT; MatTek)
- Tissue lot number: 25875

TEST FOR DIRECT MTT REDUCTION
- to check the non-specific MTT-reducing capability of the test item 25 mg of the test item were mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator (5 % CO2, 95 % RH).
- untreated MTT medium was used as control.
- if the mixture turns blue/purple, the test item is presumed to have reduced MTT and the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using freeze-killed tissues.

TEST FOR COLOUR INTERFERENCE
- to check the colouring potential of the test item 25 mg of the test item were mixed per 300 µL aqua dest. and per 300 µL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min (5 % CO2, 95 % RH).
- the mixture showed no colouring detected by unaided eye assessment. So the additional functional test with viable tissues and the quantitative corrections were not necessary.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 ± 1 minutes followed by incubation at room temperature until the 60 ± 1 minute treatment period was completed
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- after the end of the treatment period the tissues were washed 15 times with DPBS.
- subsequently, the inserts were submerged three times in DPBS and shaken to remove rests of the test item.
- then inserts were rinsed once from the inside and the outside with sterile DPBS.
- inserts were placed in prepared 6-well plates containing pre-warmed fresh assay medium per well.
- plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing fresh assay medium and incubated for additional 18 ± 2 h.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/well)
- Incubation time: 3 hours ± 5 minutes
- Extraction of formazan: after the MTT incubation period, the tissues were rinsed three times with DPBS and allowed to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature at least for 2 hours with shaking on a plate shaker.
Before using the extracts, the plate had been shaken for at least 15 minutes on a plate shaker and the inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2 x 200 µL aliquots per each tissue were transferred into a 96-well plate. Optical density (OD) was measured with a filter band without reference wavelength in a plate spectrophotometer using isopropanol as a blank.
- Wavelength: 570 nm
- Filter bandwidth: maximum ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1-virus, Hepatitis B virus and Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. This value corresponded to 100 % tissue viability in the current test. For each individual tissue treated with the test item or the positive control, the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [(mean ODtest item / positive control) / ODmean of negative control] * 100
For the test item and the positive control the mean relative viability ± relative standard deviation of the three individual tissues were calculated and used for classification according to the following prediction model:
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with DPBS. The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS “Category 2”), if the tissue viability after exposure and post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS categories 1 and 2 and decide on the final classification of the test substance. The test substance may be considered as non-irritant to skin in accordance with regulation EC 1272/2008 and UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is more than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg (39 mg/cm²) of the test item
Firstly, 25 µL of the vehicle were applied to the epidermal surface in order to improve the contact between the powder and the epidermis. Afterwards, 25 mg (39 mg/cm²) of the test item were applied directly atop the EpiDermTM tissue using an application spoon avoiding compression of the test item. The test item was spread to match size of the tissue by using a bulb-headed Pasteur pipette.

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL of the vehicle

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of 5 % SDS solution

Duration of treatment / exposure:

60 ± 1 minute

Duration of post-treatment incubation (if applicable):

approx. 42 hours

Number of replicates:

triplicates

Irritation / corrosion parameter:

% tissue viability

Remarks:

(mean)

Value:

104.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

TEST FOR MTT INTERFERENCE
The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

TEST FOR COLOUR INTERFERENCE
The mixture of 25 mg of the test item per 300 μl aqua dest. and/or per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (value: 1.845).
- Acceptance criteria met for positive control: mean relative tissue viability (% negative control) of the positive control was ≤ 20% (4.1 %)
- Acceptance criteria met for variability between replicate measurements: standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.6 % - 6.2 %).
- The absorbance values were not below historically established boundaries.

Please also refer to the field "An other information on results incl. tables" below.

Table1:  Result of the Test Item 2-ethylhexanoic acid, cerium salt

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.717	1.912	1.872	0.106	0.124	0.125	1.906	1.837	2.054
	1.743	1.895	1.929	0.107	0.125	0.124	1.878	1.820	2.036
Mean Absolute OD570	1.845****			0.119			1.922
OD570(Blank Corrected)	1.673	1.868	1.828	0.062	0.080	0.081	1.862	1.793	2.010
	1.699	1.851	1.885	0.063	0.081	0.081	1.834	1.776	1.992
Mean OD570of the Duplicates (Blank Corrected)	1.686	1.860	1.857	0.063	0.081	0.081	1.848	1.785	2.001
Total Mean OD570of 3 Replicate Tissues (Blank Corrected)	1.801*			0.075			1.878
SD of Mean OD570of the Replicates (Blank Corrected)	0.099			0.010			0.111
Relative Tissue Viability [%]	93.6	103.3	103.1	3.5	4.5	4.5	102.6	99.1	111.1
Mean Relative Tissue Viability [%]	100.0			4.1**			104.3
SD of Relative Tissue Viability [%]	5.5***			0.6***			6.2***
CV [% Viabilities]	5.5			13.9			5.9

^* Blank-corrected mean OD_{570 nm}of the negative control corresponds to 100% absolute tissue viability.

^** Mean relative tissue viability of the three positive control tissues is ≤ 20%.

^*** Standard deviation (SD)of relative tissue viabilityobtained from the three concurrently tested tissuesfor test item, positive and negative controlis ≤ 18%

****Mean absolute OD₅₇₀ of the negative control tissues is ≥0.8 and ≤2.8.

Table2:  Historical Data

	Mean Absolute OD570±30nmNC	MeanAbsoluteOD570±30nmPC	Mean Relative Viability [%] PC	SD Viability [%] NC, PC, TI
Mean	1.861	0.114	3.7	4.4
SD	0.247	0.033	1.5	4.1
Range of LCL – UCL	1.367 – 2.355	0.048 – 0.181	0.7 – 6.8	0.0 – 12.5
n	25	25	25	117

LCL:      Lower control limit (95%, mean – 2*SD)

UCL:      Upper control limit (95%, mean + 2*SD)

n:            number of control values

Historical data were generated in 2017.

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the reported conditions of the human skin model test, 2-ethylhexanoic acid, cerium salt is not irritant to the skin (No Category) according to Regulation (EC) No. 1272/2008 and UN GHS.

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:

2018-02-14

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)

Version / remarks:

2017-10-09

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-06-05

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in a sealed container

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: test item was crushed to a fine powder using a mortar and a pestle.

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: abattoir A. Moksel AG, Buchloe, Germany
- Storage, temperature and transport conditions of ocular tissue: fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories.
- Time interval prior to initiating testing: immediately after arrival of the eyes, cornea preparation was initiated and was used for BCOP testing on the same day.

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): about 750 mg of the test item
Since it was not possible to get a solution with the test item, it was administered directly and moistened with a drop of physiological saline (0.9% NaCl).

Duration of treatment / exposure:

4 hours

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

not required

Number of animals or in vitro replicates:

Number of bovine corneae per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- eyes were examined for defects and any defective eyes were discarded. Eyes with scratches or any kind of opacity were not used.
- tissue surrounding the eyeball was pulled away and the cornea was excised.
- isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with pre-warmed MEMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete MEMI).
- corneas were incubated for one hour at 32 ± 1 °C for equilibration in an air incubator.

QUALITY CHECK OF THE ISOLATED CORNEAS
- after the equilibration period, the medium was removed from both chambers and replaced with fresh complete MEM.
- an initial measurement was performed on each of the corneas using the opacitometer.
- three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas.
- the illuminance of each cornea was read and recorded.
- only corneas that had an initial illuminance reading I > I0/1.1651 lux (an equivalent to the opacity threshold of 7 as listed in OECD 437) were used for the assay.

APPLICATION DOSE AND EXPOSURE TIME
- medium was removed from the anterior chamber and replaced with the test item or control.
- 750 µL of the control substances (closed-chamber method) and enough test item to completely cover the cornea (about 750 mg, open-chamber method) were introduced into the anterior chamber.
- after 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed.

REMOVAL OF TEST SUBSTANCE/CONTROL SUBSTANCES
- epithelium was washed more than three times with MEM (containing phenol red), since the corneas showed small test item residues, which could not be removed.
- the cornea was finally rinsed with complete MEM (without phenol red).

METHODS FOR MEASURED ENDPOINTS:
- anterior chamber was refilled with complete MEM and an illuminance measurement was performed.
- corneas were visually examined for tissue peeling, residual test chemical and non-uniform opacity patterns and observation were recorded.
- after the illuminance measurement was performed, the medium was removed from both chambers of the holder.
- posterior chamber was refilled with fresh complete MEMI.
- 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C in horizontal position.
- then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

Evaluation of the opacity:
- the following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer:
Opacity = ((I0/I) - b)/ a
with a = 0.025 and b = 0.9894
- value I0 is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated periodically. This I0 value was than calculated to the respective data of the opacitometer and the data according to guideline (opacity < 7). So the initial illuminance could be calculated and corneas below this value were discarded.
- change in opacity for each cornea (test item, positive and negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values of test item treated cornea or positive control were corrected by subtracting from each the average change in opacity observed for the negative-control corneas to obtain a corrected opacity. The mean corrected opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.

Evaluation of the permeability:
- mean OD490 for the blank cuvettes was calculated.
- mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490).
- any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor.
- final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
- mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula was used to determine the in vitro irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)
To determine the IVIS of the positive control and the test item, the corrected opacity and OD490 values are used.

ACCEPTABILITY CRITERIA
- the BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
- the negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Irritation parameter:

in vitro irritation score

Remarks:

(mean)

Run / experiment:

Experiment 1

Value:

2.13

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

not valid

Remarks on result:

other: The in vitro irritation score obtained with the positive control did not fall within the two standard deviations of the current historical mean. Therefore, the assy was not considered to be valid and the experiment was repeated (Experiment 2).

Irritation parameter:

in vitro irritation score

Remarks:

(mean)

Run / experiment:

Experiment 2

Value:

2.58

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

EXPERIMENT 1:

- Measurement after treatment:
All 3 corneas treated with 2-ethylhexanoic acid, cerium salt showed very slight opacity of the tissue.

- Acceptance of results:
- Acceptance criteria met for negative control: the negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
- Acceptance criteria met for positive control: the in vitro irritation score obtained with the positive control did not fall within the two standard deviations of the current historical mean. Therefore, the assy was not considered to be valid and the experiment was repeated.

EXPERIMENT 2:

- Visual Observation after treatment:
None of the 3 corneas treated with 2-ethylhexanoic acid, cerium salt showed any opacity of the tissue. 2 corneas showed small test item residues that could not be removed by non-invasive methods, such as additional rinsing. 2 test item treated corneas showed small test item residues, that could not be removed by non-invasive methods, such as additional rinsing. An interference of residual test material with the opacity measurement is not assumed, since the residues were very small and the qualitative visual appraisal of the test item treated corneas showed no opacity of the tissue, so that a possible impact on the opacity measurement can be disregarded. Furthermore, relative to the negative control, the test item caused no increase of corneal opacity and permeability in all 3 corneas. In conclusion, the residual material has no effect on the result.

- Measurement after treatment:
Relative to the negative control, the test item caused no increase of corneal opacity and permeability in all 3 corneas.

- Acceptance of results:
The test is valid since the IVIS of the positive control falls within two standard deviations of the current historical mean and opacity and permeability of the negative control are less than the respective established upper limits for background opacity and permeability.

Please also refer for results to the field "Any other information on results incl. tables" below

EXPERIMENT 2 valid:

Table 1:  Opacity

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1	Negative Control	0.19	0.97	0.79
2		0.19	0.97	0.79
3		0.12	0.66	0.54
MV		0.16	0.87	0.71
4	Positive Control	1.40	99.43	98.03	97.33
5		1.43	79.29	77.86	77.15
6		1.01	105.47	104.46	103.75
MV		1.28	94.73	93.45	92.74
7	Test Item	-0.18	1.04	1.23	0.52
8		-1.24	5.61	6.85	6.15
9		0.73	2.09	1.36	0.65
MV		-0.23	2.92	3.15	2.44
MV = mean value

Table 2:  Permeability

Cornea No.	Test Item	OD490	Corrected OD490 Value
1	Negative Control	0.006
2		0.005
3		0.005
MV		0.005
4	Positive Control	2.715	2.710
5		3.240	3.235
6		5.280	5.275
MV		3.745	3.740
7	Test Item	0.013	0.008
8		0.009	0.004
9		0.022	0.017
MV		0.015	0.009
MV = mean value

 

Table 3:  In Vitro Irritation Score

Cornea No.	Test Item	Corrected Opacity Value	Corrected OD490 Value	IVIS
1	Negative Control	0.79	0.006
2		0.79	0.005
3		0.54	0.005
MV		0.71	0.005	0.79
4	Positive Control	97.33	2.710
5		77.15	3.235
6		103.75	5.275
MV		92.74	3.740	148.84
7	Test Item	0.52	0.008
8		6.15	0.004
9		0.65	0.017
MV		2.44	0.009	2.58
MV = mean value

Table 4: Historical mean In Vitro Irritation Score of the Positive Control (Imidazole 20%) from February 2015 until May 2018

	IVIS Positive Control - Imidazole 20 %
Mean Value (MV)	123.98
Standard Deviation (SD)	17.69
MV- 2xSD	88.60
MV+2xSD	159.37
Number of Replicates providing Historical Mean: 36

Positive controls are updated after every single experiment or at least every 3 months.

Table 5: Historical Data on Opacity and Permeability of the Positive Control (Imidazole 20%) from August 2017 until May 2018

Replicates Providing Historical Mean	Cornea No.	Opacity		Permeability		IVIS
		Change of Opacity Value	Corrected Opacity Value	OD490 Value	Corrected OD490 Value
1	4	122.785	121.861	0.662	0.624	133.420
	5	117.173	116.249	1.220	1.182
	6	102.655	101.731	2.260	2.222
2	4	108.553	106.381	1.473	1.450	123.050
	5	79.491	77.319	2.465	2.442
	6	83.618	81.446	3.065	3.042
3	4	55.644	56.308	2.200	2.189	92.540
	5	71.511	72.175	1.348	1.337
	6	65.148	65.812	2.040	2.029
4	4	68.39	67.72	2.400	2.383	112.690
	5	70.88	70.21	2.810	2.793
	6	94.23	93.56	1.945	1.928
5	4	70.53	70.35	1.296	1.284	102.440
	5	78.68	78.50	1.363	1.351
	6	91.69	91.51	1.840	1.828
6	4	95.54	94.92	1.478	1.467	114.650
	5	83.58	82.96	1.500	1.489
	6	91.11	90.49	2.095	2.084
7	4	89.35	88.86	2.855	2.838	149.420
	5	117.36	116.87	2.215	2.198
	6	130.15	129.66	2.505	2.488
8	4	80.99	80.24	2.140	2.127	120.110
	5	78.40	77.65	3.070	3.057
	6	76.27	75.51	3.290	3.277
9	4	98.03	97.33	2.715	2.710	148.838
	5	77.86	77.15	3.240	3.235
	6	104.46	103.75	5.280	5.275
Mean Value (MV)		89.040	88.390	2.251	2.234	121.906
Standard Deviation (SD)		18.814	18.573	0.916	0.919	19.360
MV- 2xSD		51.412	51.244	0.419	0.396	83.187
MV+2xSD		126.667	125.536	4.082	4.073	160.626

Table 6: Historical meanIn Vitro Irritation Score of the Negative Control (NaCl 0.9 %) from February 2015 until May 2018

	IVIS Negative Control - NaCl 0.9 %
Mean Value (MV)	1.09
Standard Deviation (SD)	0.76
MV- 2xSD	-0.44
MV+2xSD	2.62
Number of Replicates providing Historical Mean: 36

Negative controls are updated after every single experiment or at least every 3 months.

Table 7: Historical Data on Opacity and Permeability of the Negative Control (NaCl 0.9 %) from August 2017 until May 2018

Replicates Providing Historical Mean	Cornae No.	Opacity	Permeability	IVIS
		Change of Opacity Value	OD490 Value
1	1	0.234	0.008	1.49
	2	1.738	0.008
	3	0.800	0.098
2	1	0.978	0.019	2.52
	2	3.920	0.022
	3	1.617	0.028
3	1	-0.149	0.009	-0.50
	2	-0.415	0.015
	3	-1.427	0.009
4	1	0.776	0.008	0.92
	2	0.808	0.022
	3	0.418	0.020
5	1	0.035	0.010	0.35
	2	0.036	0.013
	3	0.466	0.012
6	1	1.030	0.017	0.79
	2	0.190	0.008
	3	0.640	0.009
7	1	0.714	0.024	0.74
	2	0.373	0.015
	3	0.371	0.012
8	1	1.034	0.013	0.94
	2	0.596	0.01
	3	0.623	0.015
9	1	0.789	0.006	0.79
	2	0.789	0.005
	3	0.543	0.005
Mean Value (MV)		0.649	0.016	0.893
Standard Deviation (SD)		0.894	0.017	0.813
MV- 2xSD		-1.140	-0.019	-0.732
MV+2xSD		2.438	0.051	2.519

 

EXPERIMENT 1 invalid:

Table 1: Opacity

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1	Negative Control	2.92	3.03	0.12
2		2.61	2.99	0.38
3		2.61	2.73	0.11
MV		2.71	2.92	0.20
4	Positive Control	3.30	221.22	217.91	217.71
5		3.62	72.19	68.57	68.37
6		3.58	81.13	77.55	77.35
MV		3.50	124.85	121.35	121.14
7	Test Item	2.27	4.89	2.61	2.41
8		1.36	4.72	3.36	3.15
9		3.07	3.78	0.71	0.50
MV		2.24	4.46	2.23	2.02

MV = mean value

Table 2: Permeability

Cornea No.	Test Item	OD490	Corrected OD490 Value
1	Negative Control	0.015
2		0.018
3		0.026
MV		0.020
4	Positive Control	2.625	2.605
5		3.210	3.190
6		2.945	2.925
MV		2.927	2.907
7	Test Item	0.026	0.006
8		0.034	0.014
9		0.021	0.001
MV		0.027	0.007
MV = mean value

Table 3: In vitro irritation score

Cornea No.	Test Item	Corrected Opacity Value	Corrected OD490 Value	IVIS
1	Negative Control	0.12	0.015
2		0.38	0.018
3		0.11	0.026
MV		0.20	0.020	0.50
4	Positive Control	217.71	2.605
5		68.37	3.190
6		77.35	2.925
MV		121.14	2.907	164.75
7	Test Item	2.41	0.006
8		3.15	0.014
9		0.50	0.001
MV		2.02	0.007	2.13
MV = mean value

Table 4: Historical mean in vitro irritation score of the positive control from February 2015 until January 2018

	IVIS Positive Control - Imidazole 20 %
Mean Value (MV)	122.57
Standard Deviation (SD)	17.33
MV- 2xSD	87.91
MV+2xSD	157.23
Number of Replicates providing Historical Mean: 33

Positive controls are updated after every single experiment or at least every 3 months.

Table 5: Historical data on opacity and permeability of the positive control (Imidazole 20 %) from August 2017 until January 2018

			Incubation: 240 min
	Number of Replicates	Cornea No.	Opacity		Permeability		IVIS
			Change of Opacity Value	Corrected Opacity Value	OD490 Value	Corrected OD490 Value
2017	1	4	122.785	121.861	0.662	0.624	133.420
		5	117.173	116.249	1.220	1.182
		6	102.655	101.731	2.260	2.222
	2	4	108.553	106.381	1.473	1.450	123.050
		5	79.491	77.319	2.465	2.442
		6	83.618	81.446	3.065	3.042
	3	4	55.644	56.308	2.200	2.189	92.540
		5	71.511	72.175	1.348	1.337
		6	65.148	65.812	2.040	2.029
	4	4	68.39	67.72	2.400	2.383	112.690
		5	70.88	70.21	2.810	2.793
		6	94.23	93.56	1.945	1.928
	5	4	70.53	70.35	1.296	1.284	102.440
		5	78.68	78.50	1.363	1.351
		6	91.69	91.51	1.840	1.828
2018	6	4	95.54	94.92	1.478	1.467	114.650
		5	83.58	82.96	1.500	1.489
		6	91.11	90.49	2.095	2.084
	Mean Value (MV)		86.178	85.528	1.859	1.840	113.132
	Standard Deviation (SD)		18.438	18.015	0.617	0.619	14.497
	MV- 2xSD		49.303	49.499	0.624	0.603	84.138
	MV+2xSD		123.053	121.558	3.094	3.078	142.126

Table 6: Historical mean in vitro irritation score of the negative control from February 2015 until January 2018

	IVIS Negative Control – NaCl 0.9 %
Mean Value (MV)	1.11
Standard Deviation (SD)	0.79
MV-2xSD	-0.47
MV+2xSD	2.70
Number of Replicates providing Historical Mean: 33

Negative controls are updated after every single experiment or at least every 3 months

Table 7: Historical data on opacity and permeability of the negative control (NaCl 0.9 %) from August 2017 until January 2018

			Incubation: 240 min
	Number of Replicates Providing Historical Mean	Cornae No.	Opacity	Permeability	IVIS
			Change of Opacity Value	OD490 Value
2017	1	1	0.234	0.008	1.49
		2	1.738	0.008
		3	0.800	0.098
	2	1	0.978	0.019	2.52
		2	3.920	0.022
		3	1.617	0.028
	3	1	-0.149	0.009	-0.50
		2	-0.415	0.015
		3	-1.427	0.009
	4	1	0.776	0.008	0.92
		2	0.808	0.022
		3	0.418	0.020
	5	1	0.035	0.010	0.35
		2	0.036	0.013
		3	0.466	0.012
2018	6	1	1.030	0.017	0.79
		2	0.190	0.008
		3	0.640	0.009
	Mean Value (MV)		0.650	0.019	0.928
	Standard Deviation (SD)		1.097	0.021	1.024
	MV- 2xSD		-1.543	-0.023	-1.120
	MV+2xSD		2.843	0.060	2.976

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance is not classified as serious eye damaging or eye irritant according to the Regulation (EC) No 1272/2008 and its subsequent amendments and corrections.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

The substance was not observed to be a skin irritant in a reliable in vitro skin irritation study according to OECD 439.

Eye irritation:

The substance was not observed to be an eye irritant in a reliable in vitro eye irritation study according to OECD 437.

Justification for classification or non-classification

Skin irritation

The substance does not possess a skin irritation potential based on an in vitro OECD 439 test and does not require classification as skin irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation

The substance does not possess an eye irritation potential based on an in vitro OECD 437 test and does not require classification as eye irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.