p-benzoquinone dioxime

Endpoint summary

Description of key information

Skin corrosion, in vitro: non-corrosive (3 -minutes viability 104% ; 1 -hour viability 85%), EPIDERM, OECD TG 431, 2018

Skin irritation, in vitro: non-irritating (15minutes exposure/42hour incubation viability = 101%), EPISKIN, OECD TG 439, 2018

Eye irritation, in vitro: non-irritating (IVIS = > 7.9 and < 21.0), BCOP, OECD TG 437, 2018

Eye irritation, in vitro: study result irritating (relative viability = 0.14% and < 60%), EPIOCULAR EIT, OECD TG 492, 2018

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19-02-2018 to 23-02-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Minor deviation in environmental conditions (humidity minimum 41% and temperature minimum 35.0°C) which does not affect the reliability of the study

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS : In Vitro Skin Corrosion: Human Skin Model Test

Deviations:

yes

Remarks:

Minor deviation in environmental conditions (humidity minimum 41% and temperature minimum 35.0°C) which does not affect the reliability of the study

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDerm Skin Model (EPI-200, Lot no.: 27958 kit F). The test consists of topical application of the test item on the skin tissue for 3-minute and 1-hour. After exposure the skin tissue is thoroughly rinsed to remove the test item followed by immediate determination of the cytotoxic (corrosive) effect.
Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment. The EpiDerm Skin Model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Preincubation:
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM.

Application of test item and rinsing:
The solid test item was applied directly on top of the skin tissue. The test item was spread to match the size of the tissue. The plates were incubated for approximately 2 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM just before the test item was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure. The skin was moistened with 25 µL Milli-Q water to ensure close contact of the test item to the tissue and 39.5 to 50.1 mg of the solid test item was added into the 6-well plates on top of the skin tissues. Negative Control: similarly treated with 50 µl Milli-Q water only. Positive control: 50 µl 8N KOH (potassium hydroxide 8 normal solution). After the exposure periods (3 minutes and 1 hour, respectively), the tissues were washed with phosphate buffered saline to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µl DMEM until 6 tissues (= one application time) were dosed and rinsed.

All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 41 - 80%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.0 - 37.3°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on testing laboratory historical data these deviations are not considered significant.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Solid: ca. 39.5 to 50.1 mg + 25 µL H2O (guideline requires 25 mg + 25 µL H20, more test item if necessary).
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl H2O
- Concentration (if solution): Not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl
- Concentration (if solution): 8N KOH(aq) (pre-diluted)

Duration of treatment / exposure:

Observations are made 3-minutes and 1-hour post-test item application

Duration of post-treatment incubation (if applicable):

The DMEM was replaced by 300 µl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol (MatTek corporation) over night at room temperature.

Number of replicates:

Duplicate; used for a 3-minute exposure to the test substance and two for a 1-hour exposure.

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 minute exposure

Value:

104

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Basis: mean. Time point: 3 minute exposure. Remarks: n=2 ; CV = 9.6% ; Score in terms of percentage of negative control.

Irritation / corrosion parameter:

% tissue viability

Remarks:

1 hour exposure

Value:

85

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Basis: mean. Time point: 1 hour exposure. Remarks: n=2 ; CV = 22% ; Score in terms of percentage of negative control.

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Not applicable.
- Colour interference with MTT: Screened prior to test. Not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.
- Range of historical values if different from the ones specified in the test guideline: Historic Control Data: Negative Control OD570: 3-minutes (n=111): 1.258 – 2615 ; 1-hour (n=110): 1.371 – 2.371. Positive Control HCD data are presented within the full study report and the concurrent PC was within the specified ranges.

Table 1. Mean absorption and tissue viability in the in vitro skin corrosion test with the test item

	3-minute application				1-hour application
	A (OD570)	B (OD570)	Mean (OD570)	SD	A (OD570)	B (OD570)	Mean (OD570)	SD
Negative control	1.579	1.706	1.643	±0.090	1.749	1.834	1.792	±0.060
Test Item	1.788	1.616	1.702	±0.122	1.718	1.337	1.527	±0.270
Positive control	0.153	0.146	0.149	±0.005	0.135	0.148	0.142	±0.009

Values are corrected for background absorption (0.0423). Isopropanol was used to measure the background absorption.

SD = Standard deviation

Duplicate exposures are indicated by A and B.

CV (%) = 100 - [(lowest OD570/highest OD570) x 100%]

Interpretation of results:

GHS criteria not met

Remarks:

EU criteria not met

Conclusions:

Under the conditions of this in vitro study, the test item is not considered to be corrosive to the skin.

Executive summary:

The study was performed to OECD TG 431 and EU Method B.40 BIS to assess the skin corrosion potential of the test item in accordance with GLP using a human three dimensional epidermal model (EpiDerm (EPI-200)). The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure. 50 μl of the undiluted test item was added (with a pipette) into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μl Milli-Q water (negative control) and with 50 μl 8N KOH (positive control), respectively. The positive control had a mean relative tissue viability of 7.6% after 1 hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. In the range of viability 20 - 100% the Coefficient of Variation between tissue replicates was less than 11%, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 112% and 103%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive. Under the conditions of this study, the test item is not corrosive in the in vitro skin corrosion test.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27-03-2018 to 16-04-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Minor deviation in environmental conditions (humidity minimum 62%) which does not affect the reliability of the study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Minor deviation in environmental conditions (humidity minimum 62%) which does not affect the reliability of the study

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiSkin RHE Small Model (Lot no.: 18-EKIN-015). The test consists of topical application of the test item on to the skin tissue for 15 minutes. After exposure the skin tissue is thoroughly rinsed to remove the test item and transferred to fresh medium. After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect is performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment. The EPISKIN Small Model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Pre-test checks for Direct MTT reduction and Colour Interference were completed.

Preincubation:
On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for 23 hours at 37°C. Maintenance medium and Assay medium were supplied by a recognised supplier (documented in the full study report).

Application of test item and rinsing:
Test item: Tissues were treated by application of 5 µl water and then 11.8 to 18.3 mg test item topically applied for a 15 minutes exposure period. Negative control: 25 µl PBS (negative control) was similarly applied. Positive control: 25 µl SDS (5% w/v) which was respread after 7 minutes contact time to maintain distribution of the SDS - for the remainder of the contact period. This was not deemed necessary in the negative control or test item definitive test group.
After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 62 - 85%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.6 - 37.3°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on testing laboratory historical data these deviations are not considered significant.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 11.8 to 18.3 mg test item topically applied moistened with 5 µl water (guideline requires at least 10 mg + 5 µl water)
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µl
- Concentration (if solution): Not applicable.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µl
- Concentration (if solution): 5% SDS (pre-diluted)

Duration of treatment / exposure:

15 ± 0.5 minutes at room temperature, after which the tissues were washed with phosphate buffered saline to remove residual test item.

Duration of post-treatment incubation (if applicable):

Subsequently the skin tissues were incubated for 42 hours at 37°C.

Number of replicates:

Triplicate; treatment and concurrent negative control and positive control groups

Irritation / corrosion parameter:

% tissue viability

Value:

101

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Basis: mean. Time point: 15 minute exposure. Remarks: n=3; SD = 5.3% ; Score in terms of percentage of negative control.

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Not applicable.
- Colour interference with MTT: Screened prior to test. Not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.
- Range of historical values if different from the ones specified in the test guideline: Historic Control Data (n=173): the mean OD of the positive control was 0.13; range 0.023 to 0.437. In this same period the mean OD of the negative control was 0.98; range 0.422 – 1.547.

Table 1. Mean absorption and tissue viability in the in vitro skin irritation test with the test substance

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)	SD	Mean tissue viability (% of control)	Standard Deviation (%)
Negative control	0.885	0.890	0.871	0.882	±0.010	100	1.1
Test item	0.875	0.861	0.948	0.895	±0.047	101	5.3
Positive control	0.082	0.058	0.098	0.079	±0.020	9.0	2.3

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

Values are corrected for background adsoption (0.0442). Isopropanol was used to measure background adsorption.

Negative control: Phosphate buffered saline (PBS)

Positive control: 5% (aq.) Sodium dodecyl sulphate (SDS)

Interpretation of results:

GHS criteria not met

Remarks:

EU criteria not met

Conclusions:

Under the conditions of this in vitro study, the test item is not considered to be irritating to the skin.

Executive summary:

The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test item in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small Model). The test was performed on a total of 3 tissues per test item together with negative and positive controls. Three tissues were treated by application of 5 µl water and then 11.8 to 18.3 mg test item topically applied for a 15 minutes exposure period, similarly 3 tissues 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. The positive control had a mean cell viability of 9.0% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 101%. The standard deviation value of the percentage viability of three tissues treated identically was less than 5.3%, indicating that the test system functioned properly. Since the mean relative tissue viability for the test item was above 50% after 15 minutes treatment it is considered to be non-irritant. Under the conditions of this study the test item is not irritant in the in vitro skin irritation test.

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:

key study

Study period:

14-02-2018 to 15-02-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

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

Qualifier:

equivalent or similar to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes

Species:

other: bovine

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse.
- Age at study initiation: not reported
- Weight at study initiation: not reported
- Housing: The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (recognised supplier) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32 ± 1

Vehicle:

physiological saline

Remarks:

As the test item is a solid, at 20% w/v suspension in physiological saline was utilised for test item group only

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 microlitres (as a 20% w/v test suspension in physiological saline)
- Concentration (if solution): 20%w/v in physiological saline

Duration of treatment / exposure:

240 ±1 minutes at 32 ± 1ºC.

Duration of post- treatment incubation (in vitro):

Not applicable. According to guideline corneas treated with solids are rinsed thoroughly at the end of the 240 minutes exposure period, but do not require further incubation.

Number of animals or in vitro replicates:

Three (3) per test item, or negative or positive controls, respectively.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. Following mounting: the anterior and posterior chambers of each BCOP holder were filled with complete Earle’s Minimum Essential Medium (cMEM) and the holders were incubated at 32 ± 1 ºC for a minimum of 1 hour. After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM.

QUALITY CHECK OF THE ISOLATED CORNEAS: The corneas were examined for defects macroscopically. Only corneas with opacity ≥ 7.0 are discarded, in accordance with the guideline.

NUMBER OF REPLICATES: 3 (Triplicate)

NEGATIVE CONTROL USED: physiological saline

SOLVENT CONTROL USED (if applicable): Not applicable.

POSITIVE CONTROL USED: 20% (w/v) Imidazole

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 240 ± 10 minutes

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: No.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the
anterior chamber and the cornea was rinsed three times with fresh MEM containing phenol red before a final rinse with complete MEM without phenol red. The anterior chamber was refilled with fresh complete MEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed.

- POST-EXPOSURE INCUBATION: Following the final opacity measurement the posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg Na-fluorescein/ml cMEM solution Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Measured through light transmission through the cornea quantitatively using an opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): Any other pertinent visual observations would be recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value). A test item that induces an In Vitro Irritancy Score >/=55.1 is defined as an ocular corrosive or severe irritant. A test item with an IVIS 3.0 and

Irritation parameter:

in vitro irritation score

Run / experiment:

mean (n = 3)

Value:

16

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: due to an IVIS > 3 and ≤ 55, no prediction on the classification for eye irritation can be made in the Bovine Corneal Opacity and Permeability test.

Remarks:

Under the GHS prediction model, further testing is required to conclude classification and labelling

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No. The corneas were translucent brown after the 240 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were turbid post treatment.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline:
1. 20% (w/v) Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the current historical control data (HCD) mean. ACTUAL: PC IVIS range of 86.5 to 211.4. Mean = 137.74.
2. Physiological saline solution was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values for bovine corneas treated with the respective negative control of the current historical control data (HCD). When testing solids the negative control limit for IVIS should be 5.3, opacity ≤ 5.2 and for permeability ≤ 0.205. ACTUAL: NC IVIS = 1.1, opacity ≤ 0.5 and permeability ≤ 0.040.

Table 1.0 - Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score #1, #2
Negative control	0.5	0.040	1.1
Positive control	121	1.517	144
Test item	15	0.035	16

#1 Calculated using the negative control mean opacity and mean permeability values

#2 Mean in vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

 

Table 2.0 - Opacity score

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity #1	Negative control corrected Final Opacity #2	Mean Final Opacity
Negative control	5.5	6.1	0.7		0.5
	5.1	5.5	0.3
	4.1	4.4	0.4
Positive control	3.9	128.2	124.4	124	121
	4.9	136.7	131.8	131
	4.3	112.4	108.1	108
Test item	4.5	26.1	21.6	21	15
	4.3	22.0	17.7	17
	4.1	11.4	7.3	6.8

#1 Final Opacity = Opacity after treatment – Opacity before treatment.

#2 Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control

#3 Calculations are made without rounding off.

 

Table 3.0 - Permeability score individual values (uncorrected)

Treatment	Dilution factor	OD490 1	OD490 2	OD490 3	Average OD	Final OD		Mean final negative control
Negative control	1	0.011	0.012	0.015	0.013		0.013	0.040
	1	0.077	0.082	0.085	0.081		0.081
	1	0.019	0.025	0.031	0.025		0.025
Positive control	1	1.473	1.472	1.489	1.478		1.478
	6	0.343	0.341	0.347	0.344		2.062
	1	1.321	1.342	1.322	1.328		1.328
Test item	1	0.028	0.029	0.033	0.030		0.030
	1	0.082	0.093	0.080	0.085		0.085
	1	0.108	0.109	0.107	0.108		0.108

#1 Calculations are made without rounding off.

 

Table 4.0 - In vitro irritancy score

Treatment	Final Opacity #1	Final OD4902	In vitro Irritancy Score #2
Negative control	0.7	0.013	0.9
	0.3	0.081	1.6
	0.4	0.025	0.7
Positive control	124	1.438	145
	131	1.824	159
	108	1.289	127
Test item	21	-0.010	21
	17	0.045	18
	6.8	0.068	7.9

#1 Positive control and test item are corrected for the negative control.

#2 In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value).

Interpretation of results:

other: Under the GHS prediction model, further testing is required to conclude classification and labelling

Remarks:

EU criteria

Conclusions:

Under the conditions of this study, due to an IVIS > 3 and ≤ 55, no prediction on the classification for eye irritation can be made in the Bovine Corneal Opacity and Permeability test.

Executive summary:

The study was performed to OECD TG 437 and EU Method B.47 to assess the irritancy potential of the test item to the eye following exposure to bovine corneas in accordance with GLP. A total of 3 corneas per treatment group were used. A volume of 750 microlitres of the 20%w/v test item suspension was placed the cornea. The negative control group similarly received physiological saline and the positive control group received neat 20% (w/v) Imidazole solution. For each group the corneas were incubated for 240 ± 10 minutes at 32 ± 1°C. After the incubation the solutions were removed and the corneas were washed with MEM with phenol red (Earle’s Minimum Essential Medium) and thereafter with cMEM. Possible pH effects of the test substance on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns. Subsequently the corneas were incubated for 90 ± 5 minutes at 32 ± 1°C with sodium fluorescein. Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 144.0 and was within two deviations of the historical positive control data mean and within the historical positive control range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The corneas treated with the test item showed opacity values ranging from 6.8 to 21.0 and permeability values ranging from -0.010 to 0.068 and in vitro irritancy scores ranged from 7.9 to 21.0. The test item induced ocular irritation through one endpoint (opacity), resulting in a mean in vitro irritancy score of 16 after 240 minutes of treatment. Under the conditions of this study, due to an IVIS > 3 and ≤ 55, no prediction on the classification for eye irritation can be made in the Bovine Corneal Opacity and Permeability test.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30-04-2018 to 15-06-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

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:

MatTek Corporation EpiOcular™ Eye Irritation Test (OCL-200-EIT)

Deviations:

no

GLP compliance:

yes

Species:

other: human-derived epidermal keratinocyte tissues

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The test method is indicated as a validated test method recommended by OECD and/or recognised internationally for the sequential testing strategy for the assessment of ocular irritation.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27433 Kit A, Certificate of Analysis provided in the full study report).

Vehicle:

unchanged (no vehicle)

Controls:

other: Three tissues were used for the positive and negative control groups.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg of the test item
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): Not applicable.
- Concentration (if solution): Not applicable.
- Lot/batch no. (if required): Not applicable.
- Purity: Not applicable.

Duration of treatment / exposure:

6 hours ± 15 minutes at 37.0 ± 1.0°C

Duration of post- treatment incubation (in vitro):

18 hours ± 15 minutes at 37°C

Number of animals or in vitro replicates:

Two tissues were used for each treatment group (test item, negative control and positive control).
Two additional replicates were used to determine Nonspecific colour in living tissues (NSCliving) due to colour intereference.

Details on study design:

- Details of the test procedure used: See below.
- RhCE tissue construct used, including batch number: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27433 Kit A, Certificate of Analysis provided in the full study report).
- Doses of test chemical and control substances used: 50 mg test item / 50 µL Milli-Q water (negative control) / 50 µL methyl acetate (positive control)
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): Replicate tissues were equilibrated (in its 24-well shipping container) to room temperature. Subsequently, tissues were transferred to 6-well plates and incubated for 20 ± 4 hours at 37°C in 1.0 mL fresh pre-warmed supplemented DMEM Assay Medium, which was refreshed after approximately 1 hour. All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 47 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.8 - 37.4°C). Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door were considered not to impact the study based on historic laboratory data. Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes. Solid test item (53.6 to 70.2 mg) was applied directly on top of the skin tissue. Two tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues with 50 µL Methyl Acetate (positive control) respectively.
After the exposure period with the test item (6 hours ± 15 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for a 25 ± 2 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C.
- Justification for the use of a different negative control than ultrapure H2O (if applicable): Not applicable.
- Justification for the use of a different positive control than neat methyl acetate (if applicable): Not applicable.
- Description of any modifications to the test procedure: Not applicable.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): Since the test item induced colour interference in aqueous conditions, two tissues were treated with test item. Instead of MTT solution these tissues were incubated with DMEM. Nonspecific colour in living tissues (NSCliving) was calculated. True tissue viability is calculated as the difference between the OD obtained with the test item treated living tissues incubated with MTT medium (ODlt_t+MTT) and the OD obtained with the test item treated living tissues incubated with medium without MTT (ODlt_t-MTT), and subsequently divided by the OD of the negative control (ODlt_u+MTT). The test item did not directly interact with MTT and therefore was a direct MTT reducer.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): Duplicate.
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm
- Description of the method used to quantify MTT formazan: TECAN Infinite® M200 Pro Plate – spectrophotometrically.
- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): Not applicable.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: According to the OECD TG 492 and GHS system.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: The absolute mean OD570 of the negative control tissues was marginally outside the laboratory historical control data range: 1.228 – 2.090. However, was within the OECD TG 492, acceptability range of OD > 0.8 and < 2.5 and/or the difference in viability was < 12% between replicates. The Positive Control gave a response within the historic control range: 17.80 – 34.18%. The current HCD was provided in the full study report.
- Complete supporting information for the specific RhCE tissue construct used: Attached to the full study report.
- Reference to historical data of the RhCE tissue construct: Full historic control data is attached to the full study report.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: The test assay is validated at the test laboratory and this information is in the public domain.
- Positive and negative control means and acceptance ranges based on historical data: See above.
- Acceptable variability between tissue replicates for positive and negative controls: < 12 %.
- Acceptable variability between tissue replicates for the test chemical: Yes.

Irritation parameter:

other: Mean tissue viability (% of negative control)

Remarks:

n=2

Run / experiment:

mean - test item

Value:

0.14

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

other: Mean tissue viability (% of negative control)

Remarks:

n=2

Run / experiment:

mean - positive control

Value:

25

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:

OTHER EFFECTS:
- Visible damage on test system: None.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The test assay is validated at the test laboratory and this information is in the public domain.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline: See 'details on study design' for further information.

Table 1.0 : Mean absorption and Mean Tissue Viability in the EPIOCULAR Test

	A (OD570)	B (OD570)	Mean (OD570)	SD	Mean tissue viability (percentage of control)	Difference between two tissues  (percentage)
Negative control	2.043	2.190	2.116	± 0.104	100	6.9
Test Item	0.004	0.002	0.003	±0.001	0.14	0.09
Positive control	0.650	0.397	0.523	± 0.179	25	12

 

Acceptability of the Assay

a) The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.

b) The mean relative tissue viability of the positive control should be < 50% relative to the negative control.

c) The difference between the % tissue viabilities of the two identically treated replicates should be < 20%.

d) The non-specific colour of the test item should be < 50% relative to the negative control OD.

All assay acceptability criteria were met.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Remarks:

EU criteria; incorporating expert judgement and/or information from preceding studies

Conclusions:

Under the conditions of this study, the test item is considered to be an eye irritant.

Executive summary:

The study was performed to assess the eye irritancy potential of the test item using the EpiOcular EIT model under OECD TG 492 guideline in accordance with GLP. The test item was topically applied for 6 hours. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. The tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The test item showed colour interference in aqueous conditions. In addition to the normal procedure, two tissue were treated with test item. Instead of MTT solution these tissues were incubated with assay medium. The non-specific colour of the test item in living tissues (%NSCliving) was 1.9% of the negative control tissues. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test item treated viable tissues with MTT assay. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 0.14%. The positive control had a mean cell viability of 25% after 6 hours ± 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptability criteria. The difference between the percentages of viability of two tissues treated identically was less than 12%, indicating that the test system functioned properly. Under the conditions of this study, since the test item relative mean viability was < 60% the test item is considered potentially eye irritant or corrosive.

Applicant assessment indicates: preceding test information from other OECD guideline methods in top-down strategy has already excluded GHS Eye Damage category 1 classification. The test item meets the GHS criteria for GHS Eye Irritation category 2.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion:

Key study: in vivo, OECD TG 431, 2018 : The study was performed to OECD TG 431 and EU Method B.40 BIS to assess the skin corrosion potential of the test item in accordance with GLP using a human three dimensional epidermal model (EpiDerm (EPI-200)). The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure. At least 25 mg test item moistened with 50 μl water was added (with a pipette) into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μl Milli-Q water (negative control) and with 50 μl 8N KOH (positive control), respectively. The positive control had a mean relative tissue viability of 7.9% after 1 hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. In the range of viability 20 - 100% the Coefficient of Variation between tissue replicates was less than 22%, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 104% and 85%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is not considered to be corrosive. Under the conditions of this study, the test item is not corrosive in the in vitro skin corrosion test.

 

Skin Irritation:

Key study : in vitro, OECD TG 439, 2018 : The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test item in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small Model). The test was performed on a total of 3 tissues per test item together with negative and positive controls. Three tissues were treated by application of 5 µl water and then 11.8 to 18.3 mg test item topically applied for a 15 minutes exposure period, similarly 3 tissues 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. The positive control had a mean cell viability of 9.0% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 101%. The standard deviation value of the percentage viability of three tissues treated identically was less than 5.3%, indicating that the test system functioned properly. Since the mean relative tissue viability for the test item was above 50% after 15 minutes treatment it is considered to be non-irritant. Under the conditions of this study the test item is not irritant in the in vitro skin irritation test.

 

Eye Irritation:

Key study : In vitro, OECD TG 437, 2018 : The study was performed to OECD TG 437 and EU Method B.47 to assess the irritancy potential of the test item to the eye following exposure to bovine corneas in accordance with GLP. A total of 3 corneas per treatment group were used. A volume of 750 microlitres of the 20%w/v test item suspension was placed the cornea. The negative control group similarly received physiological saline and the positive control group received neat 20% (w/v) Imidazole solution. For each group the corneas were incubated for 240 ± 10 minutes at 32 ± 1°C. After the incubation the solutions were removed and the corneas were washed with MEM with phenol red (Earle’s Minimum Essential Medium) and thereafter with cMEM. Possible pH effects of the test substance on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns. Subsequently the corneas were incubated for 90 ± 5 minutes at 32 ± 1°C with sodium fluorescein. Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 144.0 and was within two deviations of the historical positive control data mean and within the historical positive control range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The corneas treated with the test item showed opacity values ranging from 6.8 to 21.0 and permeability values ranging from -0.010 to 0.068 and in vitro irritancy scores ranged from 7.9 to 21.0. The test item induced ocular irritation through one endpoint (opacity), resulting in a mean in vitro irritancy score of 16 after 240 minutes of treatment. Under the conditions of this study, due to an IVIS > 3 and ≤ 55, no prediction on the classification for eye irritation can be made in the Bovine Corneal Opacity and Permeability test.

 

Key study : In vitro, OECD TG 492, 2018 : The study was performed to assess the eye irritancy potential of the test item using the EpiOcular EIT model under OECD TG 492 guideline in accordance with GLP. The test item was topically applied for 6 hours. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. The tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The test item showed colour interference in aqueous conditions. In addition to the normal procedure, two tissue were treated with test item. Instead of MTT solution these tissues were incubated with assay medium. The non-specific colour of the test item in living tissues (%NSCliving) was 1.9% of the negative control tissues. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test item treated viable tissues with MTT assay. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 0.14%. The positive control had a mean cell viability of 25% after 6 hours ± 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptability criteria. The difference between the percentages of viability of two tissues treated identically was less than 12%, indicating that the test system functioned properly. Under the conditions of this study, since the test item relative mean viability was < 60% the test item is considered potentially eye irritant or corrosive.

 

Weight of Evidence: The test item utilising guideline methods in top-down strategy indicates exclusion from GHS Eye Damage category 1 classification due to BCOP IVIS < 55.0. The test item meets the GHS criteria for GHS Eye Irritation category 2 due to EPIOCUAR EIT relative mean viability < 60%.

 

Respiratory Irritation:

No data available.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for dermal irritation.

 

The substance meets classification criteria under Regulation (EC) No 1272/2008 for eye irritation: category 2: H319: causes serious eye irritation

Weight of Evidence: Within eye irritation, the test item utilising guideline methods in top-down strategy indicates exclusion from GHS Eye Damage category 1 classification due to BCOP IVIS < 55.0. The test item meets the GHS criteria for GHS Eye Irritation category 2 due to EPIOCUAR EIT relative mean viability < 60%.