1-Naphthol, reaction products with formaldehyde

Endpoint summary

Description of key information

Skin Corrosion (Eurlings, 2018)

Under the conditions of this study no prediction of skin corrosion potential can be made.

Eye Corrosion (Eurlings, 2018)

Under the conditions of this study, the test material induces serious eye damage.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 January 2018 to 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:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test".

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended test system in international guidelines.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Lot no.: 27912 Kit E and Kit F.
- Source: MatTek Corporation, Ashland MA, U.S.A.
- The 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 organised 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.

TEST FOR COLOUR INTERFERENCE BY THE TEST MATERIAL
The test material was checked for possible colour interference before the study was started. Some non-coloured test materials may change into coloured materials in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, 50 μL of the test material or 50 μL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0 °C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.

TEST FOR REDUCTION OF MTT BY THE TEST MATERIAL
The test material was checked for possible direct MTT reduction before the study was started. To assess the ability of the test material to reduce MTT, 50 μL of the test material or 50 μL Milli-Q water as a negative control were added to 1 mL MTT solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0 °C. At the end of the exposure time it was checked if a blue / purple colour change or a blue / purple precipitate was observed.

TEST SYSTEM SET-UP
- Tissues: 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.
- Freeze-killed tissues (EPI-200, Lot no.: 27636 Kit L and 27161 Kit J): Living epidermis was transferred to a freezer (≤-15 °C), thawed, and then again transferred to (≤-15 °C). The freeze-killed epidermis was stored at ≤ -15 °C until use. Freeze-killed tissues were thawed by placing them for 1 hour at room temperature in a 6 well plate on 0.9 mL DMEM. Further use of killed tissues was similar to living tissues.

APPLICATION AND TREATMENT
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well. The level of the DMEM was just beneath the tissue. The plates were incubated for approximately 2.5 hours at 37.0 ± 1.0 °C. The medium was replaced with fresh DMEM just before the test material was applied. The test was performed on a total of 4 tissues per test material together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test material and two for a 1-hour exposure. Fifty μL of the undiluted test material was added into the 6-well plates on top of the skin tissues.
In addition, since the test material induced colour interference in aqueous conditions, two tissues were treated with test material for 3 minutes and two tissues for 1-hour. Instead of MTT solution these tissues were incubated with DMEM. In addition, since the test material reacted with the MTT medium, two freeze-killed tissues were treated with test material and two freeze-killed non treated tissues were used per exposure time for the cytotoxicity evaluation with MTT.
Furthermore, since the test material was identified as MTT reducer and caused colour interference, a third set of adapted controls was required. In this control, the test material was applied to two killed tissue replicates per exposure time which underwent the entire testing procedure but were incubated with assay medium instead of MTT solution during the MTT assay.
For the negative and positive controls, 2 tissues were treated with 50 μL Milli-Q water (negative control) and 2 tissues were treated with 50 μL 8N KOH (positive control) for both the 3-minute and 1-hour time point.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minutes at room temperature, 1 hour at 37 °C
- Temperature of post-treatment incubation: 37 °C with MTT

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test material. 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.

CELL VIABILITY MEASUREMENT
- 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 over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

NUMBER OF REPLICATE TISSUES: 2

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

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

Step 1:
< 50 % after 3 minute exposure = corrosive
≥ 50 % after 3 minute exposure AND < 15 % after 1 hour exposure = corrosive
≥ 50 % after 3 minute exposure AND ≥ 15 % after 1 hour exposure = Non-corrosive
Step 2 (for substances/mixtures identified as Corrosive in step 1):
< 25 % after 3 minute exposure = Optional Sub-category 1A
≥ 25 % after 3 minute exposure = A combination of optional Sub-categories 1B and 1C

ANALYSIS
Calculation of Cell Viability
Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed.
The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).

ODc = ODraw – ODbl

The OD value representing 100 % cell viability is the average OD of the negative controls (ODlt_u+MTT).
The % Viability for each sample and positive control is calculated as follows:

%Viability = (ODc/mean ODlt_u+MTT) * 100

Colouring Test Materials
Nonspecific colour in living tissues (NSCliving) was calculated. NSCliving is the mean OD of the treated living tissues without MTT reagent (ODlt_t-MTT) expressed as percentage of the mean of the negative control tissues (ODlt_u+MTT).

%NSCliving = [ODlt_t-MTT / ODlt_u+MTT] * 100

True tissue viability is calculated as the difference between the OD obtained with the test material treated living tissues incubated with MTT medium (ODlt_t+MTT) and the OD obtained with the test material treated living tissues incubated with medium without MTT (ODlt_t-MTT), and subsequently divided by the OD of the negative control (ODlt_u+MTT).

OD = ODlt_t+MTT – ODlt-t-MTT

% Viability = (OD / mean ODlt_u+MTT) * 100.

Since the %NSCliving ≤ 0.0, there is no need to correct for colour interference of the test material.

MTT Interacting Test Materials
Nonspecific MTT reduction (NSMTT) was calculated. NSMTT is the difference between the mean OD of the untreated freeze-killed tissues (ODkt_u+MTT) and test material treated freeze-killed tissues (ODkt_t+MTT) expressed as percentage of the mean of the negative control tissues (ODlt_u+MTT).

%NSMTT = [(ODkt_t+MTT – ODkt_u+MTT)/ mean ODlt_u+MTT] * 100

True tissue viability is calculated as the difference between the living test material treated tissues
incubated with MTT medium (ODlt_t+MTT) and the difference between ODkt_t+MTT and ODkt_u+MTT.

OD= [ODlt_t+MTT – (ODkt_t+MTT-ODkt_u+MTT)]

%Viability = [OD/ mean ODlt_u+MTT] * 100

Since the %NSMTT ≤ 0.0, there is no need to correct for interference of the test material.

Colouring and MTT Interacting Test Materials
Nonspecific colour in freeze-killed tissues (NSCkilled) was calculated. NSCkilled is the mean OD of the test material treated killed tissues without MTT reagent (ODkt_t-MTT) expressed as percentage of the mean of the negative control tissues (ODlt_u+MTT).

%NSCkilled = (ODkt_t-MTT) / ODlt_u+MTT) * 100

True tissue viability is calculated as the OD obtained with the test material treated living tissues incubated with MTT medium (ODlt_t+MTT), minus the OD obtained with the test material treated living tissues incubated with medium without MTT (ODlt_t-MTT), minus the difference between the mean OD of the untreated freeze-killed tissues (ODkt_u+MTT) and test material treated freeze-killed tissues (ODkt_t+MTT) plus the mean OD of the test material treated killed tissues without MTT reagent (ODkt_t-MTT), subsequently divided by the OD of the negative control (ODlt_u+MTT).

OD = ODlt_t+MTT– ODlt_t-MTT – (ODkt_t+MTT-ODkt_u+MTT) + ODkt_t-MTT

%Viability = (OD / ODlt_u+MTT) * 100.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL
- Concentration: Undiluted. No correction was made for hte purity/composition of the test material.

NEGATIVE CONTROL
- Amount(s) applied: 50 μL

POSITIVE CONTROL
- Amount(s) applied: 50 μL
- Concentration: 8.0 N

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours with MTT

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

34

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure

Value:

3.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- The test material was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test material to MTT medium. Because a colour change was observed in aqueous conditions and by adding MTT-medium it was concluded that the test material did interact with the MTT endpoint.
- In addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test material and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each time point. The non-specific reduction of MTT by the test material was 79 and 89 % of the negative control tissues after 3 minutes and 1 hour respectively.
- In addition to the normal 3-minute and 1-hour procedure, two tissues were treated with test material. Instead of MTT solution these tissues were incubated with DMEM. The colour interference by the test material was 3.85 and 32 % of the negative control tissues after 3 minutes and 1 hour respectively.
- For the true tissue viability to be calculated, in addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test material were measured but instead of MTT solution these tissues were incubated with DMEM. The nonspecific colour in freeze-killed tissues by the test material was 20% and 28% of the negative control tissues after 3 minutes and 1 hour respectively.
- Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 34 and 3.6 % respectively.
- The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 7.6 %.
In the range of 20 – 100 % viability the Coefficient of Variation between tissue replicates was < 10 % , indicating that the test system functioned properly.
Because the non-specific reduction of MTT by the test material was 79 and 89 % of the negative control tissues after 3 minutes and 1 hour respectively, which is far above the acceptability criteria of 30 %, this test is considered unsuitable for this test material and therefore no prediction can be made.

Table 1: Mean Absorption in the in vitro Skin Corrosion Test

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.906	2.113	2.010	±	0.146	1.934	2.100	2.017	±	0.118
Test Material^(1)	0.724	0.662	0.693	±	0.044	0.011	0.133	0.072	±	0.087
Positive control	0.184	0.170	0.177	±	0.010	0.162	0.144	0.153	±	0.013

SD = Standard deviation

Duplicate exposures are indicated by A and B.

(1) The test material values are corrected for the non-specific MTT reaction and color interference (79 and 89 for the MTT reaction and 3.85 and 32 for the color interference at the 3 minute and 1 hour treatment, respectively).

Table 2: Mean Tissue Viability in the in vitro Skin Corrosion Test

Treatment	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative Control	100	100
Test Material	34	3.6
Positive Control	8.8	7.6

Interpretation of results:

other: No prediction can be made

Conclusions:

Under the conditions of this study no prediction of skin corrosion potential can be made.

Executive summary:

The skin corrosion potential of the test material was investigated in accordance with the standardised guidelines OECD 431 and EU Method B.40.bis, under GLP conditions.

The objective of this study was to evaluate the test material for its ability to induce skin corrosion on a human three dimensional epidermal model. The possible corrosive potential of the test material was tested through topical application for 3 minutes and 1 hour. The test material was applied undiluted (50 µL) directly on top of the skin tissue. 

The positive control had a mean relative tissue viability of 7.6 % after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. In the range of 20 – 100 % viability the Coefficient of Variation between tissue replicates was < 10 %, indicating that the test system functioned properly.

Because a colour change was observed in aqueous conditions and by adding MTT-medium it was concluded that the test material did interact with the MTT endpoint.

In addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test material and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each time point. The non-specific reduction of MTT by the test material was 79 and 89 % of the negative control tissues after 3 minutes and 1 hour respectively.

In addition to the normal 3-minute and 1-hour procedure, two tissues were treated with test material. Instead of MTT solution these tissues were incubated with DMEM. The colour interference by the test material was 3.85 and 32 % of the negative control tissues after 3 minutes and 1 hour respectively. 

For the true tissue viability to be calculated, in addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test material were measured but instead of MTT solution these tissues were incubated with DMEM. The nonspecific colour in freeze-killed tissues by the test material was 20 and 28 % of the negative control tissues after 3 minutes and 1 hour respectively. 

Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 34 and 3.6 %, respectively. Because the non-specific reduction of MTT by the test material was 79 and 89 % of the negative control tissues after 3 minutes and 1 hour respectively, which is far above the acceptability criteria of 30 %, this test is considered unsuitable for this test material and therefore no prediction can be made.

Under the conditions of this study no prediction of skin corrosion potential can be made.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Data waiving:

study technically not feasible

Justification for data waiving:

other:

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:

17 January 2018 to 08 February 2018

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:

2013

Deviations:

no

GLP compliance:

yes

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: Bovine eyes were used as soon as possible after slaughter.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularisation by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
- Indication of any antibiotics used: None reported

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration: Neat test material

Duration of treatment / exposure:

10 ± 1 minutes

Duration of post- treatment incubation (in vitro):

120 ± 10 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1 % (v/v) L-glutamine and 1 % (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) 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.

QUALITY CHECK OF THE ISOLATED CORNEAS
- After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
- Three corneas were selected at random for each treatment group.

VEHICLE CONTROL USED: Physiological saline

POSITIVE CONTROL USED: Ethanol

APPLICATION DOSE AND EXPOSURE TIME: 750 µL for 10 ± 1 minutes at 32 ± 1 °C.

TREATMENT METHOD
- The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control or test material was introduced onto the epithelium of the cornea.
- The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1 °C.

REMOVAL OF TEST SUBSTANCE
- 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. Possible pH effects of the test material on the corneas were recorded.
- The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1 °C.

METHODS FOR MEASURED ENDPOINTS:
- After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
- The opacity value (measured with the device OP-KIT) was calculated according to:

Opacity = [(I0 / I) - 0.9894] / 0.0251

With I0 being the empirically determined illuminance through a cornea holder but with windows and medium and I being the measured illuminance through a holder with cornea.
-The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test material or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test material or positive control treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
- Corneal permeability: Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1 °C.
- After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader. Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
- The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test material was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
- 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)

Additionally the opacity and permeability values were evaluated independently to determine whether the test material induced irritation through only one of the two endpoints.

DECISION CRITERIA
- The IVIS cut-off values for identifying the test materials as inducing serious eye damage (UN GHS Category 1) and test materials not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are: In vitro score range: ≤ 3 = UN GHS No Category; > 3 but ≤ 55 = No prediction can be made; and >55 = UN GHS Category 1

ACCEPTABILITY OF THE ASSAY
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

10 085

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

RESULTS
The individual in vitro irritancy scores for the negative controls ranged from -2.3 to -0.7.
The individual positive control in vitro irritancy scores ranged from 37 to 53. The corneas treated with the positive control material were turbid after the 10 minutes of treatment.
The corneas treated with the test material showed opacity values ranging from 3902 to 19677 and permeability values ranging from 2.237 to 4.401. Due to the colouring of the test material, the corneas could not be visually inspected for dissimilar opacity patterns. No pH effect of the test material was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 3968 to 19721 after 10 minutes of treatment with the test material.

DISCUSSION
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 (Ethanol) was 46 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
The test material induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 10085 after 10 minutes of treatment.

Table 1: Opacity, Permeability and In Vitro Scores

Treatment		Opacity	Permeability (Corrected)	IVIS
Negative control	1	-2.3	-0.005	2.3
	2	-1.1	-0.001	-1.1
	3	-0.7	-0.004	-0.7
	Mean	-1.3	-0.003	-1.4
Positive control	1	16	2.405	53
	2	15	1.436	37
	3	15	2.159	47
	Mean	16	2.000	46
Test material	1	19677	2.941	19721
	2	3902	4.401	3968
	3	6531	2.237	6565
	Mean	10037	3.193	10085

Interpretation of results:

other: EU Criteria: Category 1 (H318 Causes serious eye damage)

Conclusions:

Under the conditions of this study, the test material induces serious eye damage.

Executive summary:

The potential of the test material to cause damage to the eye was investigated in accordance with the standardised guideline OECD 437, under GLP conditions.

The objective of this study was to evaluate the eye hazard potential of the test material as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

The test material was tested through topical application to isolated bovine corneas for 10 minutes. The test item was applied as it is (750 μL) directly on top of the corneas.

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 (Ethanol) was 46 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The test material induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 10085 after 10 minutes of treatment. Since the test material induced an IVIS ≥ 55, it is concluded that the test material is classified as Category 1.

Under the conditions of this study, the test material induces serious eye damage.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion (Eurlings, 2018)

The skin corrosion potential of the test material was investigated in accordance with the standardised guidelines OECD 431 and EU Method B.40.bis, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of this study was to evaluate the test material for its ability to induce skin corrosion on a human three dimensional epidermal model. The possible corrosive potential of the test material was tested through topical application for 3 minutes and 1 hour. The test material was applied undiluted (50 µL) directly on top of the skin tissue. 

The positive control had a mean relative tissue viability of 7.6 % after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. In the range of 20 – 100 % viability the Coefficient of Variation between tissue replicates was < 10 %, indicating that the test system functioned properly.

Because a colour change was observed in aqueous conditions and by adding MTT-medium it was concluded that the test material did interact with the MTT endpoint.

In addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test material and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each time point. The non-specific reduction of MTT by the test material was 79 and 89 % of the negative control tissues after 3 minutes and 1 hour respectively.

In addition to the normal 3-minute and 1-hour procedure, two tissues were treated with test material. Instead of MTT solution these tissues were incubated with DMEM. The colour interference by the test material was 3.85 and 32 % of the negative control tissues after 3 minutes and 1 hour respectively. 

For the true tissue viability to be calculated, in addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test material were measured but instead of MTT solution these tissues were incubated with DMEM. The nonspecific colour in freeze-killed tissues by the test material was 20 and 28 % of the negative control tissues after 3 minutes and 1 hour respectively. 

Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 34 and 3.6 %, respectively. Because the non-specific reduction of MTT by the test material was 79 and 89 % of the negative control tissues after 3 minutes and 1 hour respectively, which is far above the acceptability criteria of 30 %, this test is considered unsuitable for this test material and therefore no prediction can be made.

Under the conditions of this study no prediction of skin corrosion potential can be made.

Skin Irritation

The in vitro skin corrosion / irritation tests are not technically possible.  Attempts to assess the skin corrosion potential were imade according to OECD 431 using a reconstructed human epidermis test, however no prediction was possible due to colour interference from the test material.  The test material is a black powder which when in solution forms a blue liquid.  As the test material showed approximately 80 % non-specific interference in the OECD 431 test it was concluded that this test and the in vitro skin irritation test are not technically possible for this substance.

Eye Corrosion (Eurlings, 2018)

The potential of the test material to cause damage to the eye was investigated in accordance with the standardised guideline OECD 437, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of this study was to evaluate the eye hazard potential of the test material as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

The test material was tested through topical application to isolated bovine corneas for 10 minutes. The test item was applied as it is (750 μL) directly on top of the corneas.

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 (Ethanol) was 46 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The test material induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 10085 after 10 minutes of treatment. Since the test material induced an IVIS ≥ 55, it is concluded that the test material is classified as Category 1.

Under the conditions of this study, the test material induces serious eye damage.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance is classified as a Category 1 eye irritant (H318 Causes serious eye damage).