(Z)-N-[2-(2-hydroxyethoxy)ethyl]-9-octadecenamide

Endpoint summary

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:

From February 13, 2017 to February 17, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B40 Bis (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch: LAB 4417; Appearance: light yellow paste
- No correction factor for purity was applied
- Stability at higher temperatures: yes, maximum temperature: 30°C

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Remarks:

Moistened with Milli-Q water to ensure close contact.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 24982
- Surface: 0.6 cm^2
- Tissues were kept in the refrigerator on the day of receivement for one day

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minute exposure: room temperature, 1 hour exposure: 36.6 - 37.0°C.
- Temperature of post-treatment incubation: 36.6 - 37.0°C.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissues were washed with phosphate buffered saline (one washing step)
- Observable damage in the tissue due to washing: no

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

NUMBER OF REPLICATE TISSUES: 4 (2 tissues for the 3 minute exposure period, 2 tissues for the 1 hour exposure period) + 1 tissue for the negative and the positive control

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

PREDICTION MODEL / DECISION CRITERIA (see table 1 in ''any other informatin on methods'')
A test substance is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test substance 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 substance is decreased below 15%.

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

ACCEPTABILITY CRITERIA:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8).
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15%.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: excess amount on tissues moistened with 25 μl Milli-Q water

NEGATIVE CONTROL
- Amount applied: 50 μl

POSITIVE CONTROL
- Amount: 50 μl

Duration of treatment / exposure:

- 3 minutes
- 1 hour

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

4 in total; 2 per exposure period

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of replicates

Value:

99

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

29%

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of replicates

Value:

72

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

9%

Other effects / acceptance of results:

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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 was between the acceptance limits (i.e., 1.382 for the 3 minute exposure period and 1.603 for the 1 hour exposure period).
- Acceptance criteria met for positive control: yes, the mean relative tissue viability following 1 hour exposure was <15% (i.e., 9%).
- Acceptance criteria met for variability between replicate measurements: yes, CV between tissue replicates was ≤30% (i.e., 30% for the 3 minute exposure period and 19% for the 1 hour exposure period).

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance is not corrosive in the in vitro skin corrosion test.

Executive summary:

A study was conducted to determine the skin corrosion potential of the test substance according to the Reconstructed Human Epidermis (RHE) Test method following OECD Guideline 431 and EU Method B.40-BIS. Ability of the test substance to induce skin corrosion on a human three dimensional epidermal model was evaluated. Two tissues of the EpiDerm (EPI-200) model were treated with the test substance for 3 min and 1 h, respectively. Skin tissue was moistened with 25 μl of Milli-Q water and an excess amount of the test substance was applied directly on top of the skin tissue. The relative mean tissue viability obtained after 3 min and 1 h treatments with test substance compared to the negative control tissues was 99 and 72%, respectively. Because the mean relative tissue viability for the test substance was not below 50% after the 3 min treatment and not below 15% after the 1 h treatment the test substance is considered to be not corrosive. After treatment with the negative control, the absorbance values were within the required acceptability criterion, showing the quality of the tissues. The positive control showed clear corrosive effects for both treatment intervals. Under the study conditions, the test substance was not corrosive in the in vitro skin corrosion test (Buskens, 2017).

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 28, 2017 to April 03, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch: LAB 4417; Appearance: light yellow paste
- No correction factor for purity was applied
- Stability at higher temperatures: yes, maximum temperature: 30°C

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

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

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model(TM)
- Tissue batch number(s): 17-EKIN-013
- Surface: 0.38 cm^2
- Expiration date: 3 April 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 35.9 - 36.9°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were washed with PBS (1 washing step)
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE: the test substance was checked for possible direct MTT reduction and color interference in a previous skin corrosion test using EpiDerm as a skin model. It was concluded that the test substance did not interfere with the MTT endpoint.
- Incubation time: 3 hours
- Measurement method: TECAN Infinite M200 Pro Plate Reader (570 nm)
- MTT concentration: 0.3 mg/mL

NUMBER OF REPLICATE TISSUES: 3 for the test substance, the negative and the positive control each.

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

CELL VIABILITY MEASUREMENT: After incubation, tissues were dried and incubated with 2 mL MTT-solution for 3 hours. After incubation, epidermis was separated from the collagen matrix and both parts were extracted with 500 μL isopropanol. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

PREDICTION MODEL / DECISION CRITERIA (see Table 1)
A test substance is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test substance is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is >50% of the mean viability of the negative controls.

ACCEPTABILITY CRITERIA
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: an excessive amount matching the size of the tissue (at least 10 mg).

NEGATIVE CONTROL
- Amount applied 25 μL

POSITIVE CONTROL
- Amount applied: 25 μL, re-spread afer 7 minutes of contact time

Duration of treatment / exposure:

15 +/- 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours; + 3 hours with MTT

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 replicates

Value:

76

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100% (SD: 5.2%)

Positive controls validity:

valid

Remarks:

3.7% (SD: 0.86%)

Other effects / acceptance of results:

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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the negative control tissues was within the laboratory historical control data range and the SD of the % viability was 5.2%.
- Acceptance criteria met for positive control: yes, the mean relative tissue viability was 3.7% and the SD of the % viability was 0.86%.
- Acceptance criteria met for variability between replicate measurements: no, the standard deviation of the three tissue replicates treated with test substance was 21% (> 18%). However, since all individual viabilities were >50% the outcome was clearly negative, this does not influence the outcome of the test.

- Since the mean relative tissue viability for the test substance was above 50% the test substance is considered to be non-irritant.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance is non-irritant in the in vitro skin irritation test.

Executive summary:

A study was conducted to determine the in vitro skin irritation potential of the test substance according to OECD Guideline 439 and EU Method B.46. The ability of the test substance to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKINSMTM)) was evaluated. 10 mg of the test substance was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 h post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test substance compared to the negative control tissues was 76%. Since the mean relative tissue viability for the test substancewas above 50% after 15 ± 0.5 minutes treatment the test substance is considered to be non-irritant. The positive control had a mean cell viability of 3.7% after 15 ± 0.5 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 standard deviation value of the percentage viability of three tissues treated identically was less than 6% for the negative and positive control, indicating that the test system functioned properly. Under the study conditions, the test substance was non-irritating in the in vitro skin irritation test (Eurlings, 2017).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

From February 07, 2017 to February 28, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see "Principles of method if other than guideline"

Principles of method if other than guideline:

Corneas were incubated for 10 ± 1 minutes at 25 °C in experiment 1 and at 28.5 °C in experiment II.
Evaluation: Since the test compound is not stable at higher temperatures (maximum temperature 30 ºC), the corneas were incubated with the test compound for 10 minutes at a temperature below 30 ºC. Positive and negative controls were in experiment 1 also incubated at the lower temperature. Ten minutes incubation at a slightly lower temperature has no effect on the results. The controls in experiment I were within the historical data control range and therefore this deviation has no effect on the study integrity.

GLP compliance:

yes

Specific details on test material used for the study:

Batch: LAB 4417; Appearance: light yellow paste
- No correction factor for purity was applied
- Stability at higher temperatures: yes, maximum temperature: 30°C

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System: bovine eyes were used as soon as possible after slaughter.
Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

No correction was made for the purity/composition of the test substance. An excessive amount of test substance that completely covered the cornea was applied.

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

First experiment: 10 ± 1 minutes at 25.0 °C (study plan deviation),
Second sexperiment: 10 ± 1 min at 28.5 °C (study plan deviation).

Number of animals or in vitro replicates:

3 replicates

Details on study design:

Preparation of Corneas:

The eyes were checked for unacceptable defects. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM. The isolated corneas were mounted in a corneal holder (one cornea per holder) (BASF) 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.

Cornea Selection and Opacity Reading:
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, 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. Three corneas were selected at random for each treatment group.

Treatment of Corneas and Opacity Measurements: Two experiments were performed.

- All experiments were performed at a temperature slightly below 30 °C since the test substance was not considered stable above 30 °C.
- The medium from the anterior compartment was removed and 750 uL of either the negative control, positive control (Ethanol) or an excessive amount of test substance was introduced onto the epithelium of the cornea.
- The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test substance over the entire cornea.

- Experiment I:
Corneas treated with the test substance, negative and positive control were in the first experiment incubated in a horizontal position for 10 ± 1 minutes at 25.0 °C (study plan deviation). A second experiment was performed since the IVIS values were spread over two categories.

- Experiment II:
The second experiment was aborted since the controls and test substance were incubated on the corneas for more than 10 min. The second experiment was repeated. Corneas treated with negative and positive control were in the repeat of the second experiment incubated in a horizontal position for 10 ± 1 minutes at 32.0 °C ± 1 °C. The test substance was incubated in a horizontal position for 10 ± 1 min at 28.5 °C (study plan deviation). After the incubation the solutions and the test substance were removed and the epithelium was washed with MEM with phenol red and thereafter with cMEM.
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. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

Application of Sodium Fluorescein:

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.

Permeability Determinations:

After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 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 (TECAN Infinite M200 Pro Plate 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 substance was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

Irritation parameter:

in vitro irritation score

Run / experiment:

Experiment I

Value:

1.37

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

Experiment II

Value:

-1.53

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of two experiments

Value:

-0.1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- 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 53 and 64. In experiment I and II, respectively 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.

Results Experiment I:

 

- The IVIS for the negative controls ranged from 1.0 to 2.9. The individual positive control IVIS ranged from 48 to 62.

- The corneas treated with the positive control substance were translucent after the 10 min of treatment. The cornea treated with the negative control were slightly translucent (but all IVIS values were within the historical control data range).

- The corneas treated with the test substance showed opacity values ranging from -0.8 to 2.3 and permeability values ranging from 0.023 to 0.060. The corneas were slightly translucent after the 10 min of treatment with the test substance. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 0.0 to 3.2 after 10 min of treatment with the test substance. Since the individual in vitro irritancy scores were spread over 2 categories (0.0, 0.9 and 3.2 respectively), the test was inconclusive and a repeat experiment was performed.

Results Experiment II:

 

- The IVIS for the negative controls ranged from -1.2 to -0.5. The individual positive control IVIS ranged from 48 to 82. The corneas treated with the positive control were turbid after the 10 min of treatment.

- The corneas treated with the test substance showed opacity values ranging from -2.2 to -1.0 and permeability values ranging from 0.003 to 0.040. The corneas were clear after the 10 min of treatment with the test substance. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -2.2 to -1.0 after 10 minutes of treatment with the test substance.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, over two experiments, the test substance did not induce ocular irritation through both endpoints with an IVIS <3 in 5 out of 6 eyes, resulting in a mean in vitro irritancy score of -0.1 (-2.2 to 3.2) after 10 minutes of treatment. Since the test substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

A study was conducted to determine the eye irritation potential of the test substance as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea according to OECD Guideline 437 (Bovine Corneal Opacity and Permeability test). Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, The Netherlands). Two experiments were performed. The cornea of a bovine eye was pre-incubated with cMEM without phenol red at 32 ± 1°C for 1 h and its opacity had been determined. The test substance (pure) was applied on cornea in a way that as much as possible of the cornea surface was covered. Subsequently cornea was incubated for 10 min at 32 ± 1°C. After removal of the test substance and 2 h post-incubation, opacity and permeability values were measured. Physiological saline was used as negative control and ethanol as positive control. The meanin vitroirritancy score was 1.4 after 10 min of treatment with the test substance. Since the individualin vitroirritancy scores were spread over 2 categories (0.0, 0.9 and 3.2 respectively) the test was inconclusive and a repeat experiment was performed. In the second experiment, the test substance did not induce ocular irritation through both endpoints, resulting in a meanin vitroirritancy score of -1.5 after 10 min of treatment. In both experiments, the negative controls responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative controls did not induce irritancy on the corneas. The meanin vitroirritancy score of the positive controls were within standard deviations of the current historical positive control means. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Under the study conditions, over two experiments, the test substance did not induce ocular irritation through both endpoints with an IVIS <3 in 5 out of 6 eyes, resulting in a meanin vitroirritancy score of -0.1 (-2.2 to 3.2) after 10 minutes of treatment. Since the test substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage (Westernik, 2017).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation in vitro:

A study was conducted to determine the in vitro skin irritation potential of the test substance according to OECD Guideline 439 and EU Method B.46. The ability of the test substance to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKINSMTM)) was evaluated. 10 mg of the test substance was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 h post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test substance compared to the negative control tissues was 76%. Since the mean relative tissue viability for the test substancewas above 50% after 15 ± 0.5 minutes treatment the test substance is considered to be non-irritant. The positive control had a mean cell viability of 3.7% after 15 ± 0.5 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 standard deviation value of the percentage viability of three tissues treated identically was less than 6% for the negative and positive control, indicating that the test system functioned properly. Under the study conditions, the test substance was non-irritating in the in vitro skin irritation test (Eurlings, 2017).

Skin irritation in vivo:

A study was conducted to determine the in vivo skin irritation potential of the test substance according to OECD Guideline 404 and OPPTS 870.2500.The substance was first tested in an in vitro skin irritation test (OECD 439) using an EPISKIN model (study number 517077). The substance was not an irritant in this test. However, based on similar substances causing skin irritation in rabbits, an in vivo test was conducted to confirm the in vitro result. In the current study, the test substance was applied at a dose of 0.5 mL to an unabraded site on a clipped area of each of three albino rabbits. The application sites were graded for indication of skin reactions at 4.5, 24, 48 72, 168 and 336 h after test substance application. There were moderate skin irritation reactions in all of the test subjects. The maximum skin irritation score was 5.0 at the 24 h observation. Under the study conditions, the primary skin irritation index was calculated to be 3.99 (Kukulinski, 2017).

Skin corrosion:

A study was conducted to determine the skin corrosion potential of the test substance according to the Reconstructed Human Epidermis (RHE) Test method following OECD Guideline 431 and EU Method B.40-BIS. Ability of the test substance to induce skin corrosion on a human three dimensional epidermal model was evaluated. Two tissues of the EpiDerm (EPI-200) model were treated with the test substance for 3 min and 1 h, respectively. Skin tissue was moistened with 25 μl of Milli-Q water and an excess amount of the test substance was applied directly on top of the skin tissue. The relative mean tissue viability obtained after 3 min and 1 h treatments with test substance compared to the negative control tissues was 99 and 72%, respectively. Because the mean relative tissue viability for the test substance was not below 50% after the 3 min treatment and not below 15% after the 1 h treatment the test substance is considered to be not corrosive. After treatment with the negative control, the absorbance values were within the required acceptability criterion, showing the quality of the tissues. The positive control showed clear corrosive effects for both treatment intervals. Under the study conditions, the test substance was not corrosive in the in vitro skin corrosion test (Buskens, 2017).

Eye irritation:

A study was conducted to determine the eye irritation potential of the test substance as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea according to OECD Guideline 437 (Bovine Corneal Opacity and Permeability test). Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, The Netherlands). Two experiments were performed. The cornea of a bovine eye was pre-incubated with cMEM without phenol red at 32 ± 1°C for 1 h and its opacity had been determined. The test substance (pure) was applied on cornea in a way that as much as possible of the cornea surface was covered. Subsequently cornea was incubated for 10 min at 32 ± 1°C. After removal of the test substance and 2 h post-incubation, opacity and permeability values were measured. Physiological saline was used as negative control and ethanol as positive control. The mean in vitro irritancy score was 1.4 after 10 min of treatment with the test substance. Since the individual in vitro irritancy scores were spread over 2 categories (0.0, 0.9 and 3.2 respectively) the test was inconclusive and a repeat experiment was performed. In the second experiment, the test substance did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -1.5 after 10 min of treatment. In both experiments, the negative controls responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative controls did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive controls were within standard deviations of the current historical positive control means. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Under the study conditions, over two experiments, the test substance did not induce ocular irritation through both endpoints with an IVIS <3 in 5 out of 6 eyes, resulting in a mean in vitro irritancy score of -0.1 (-2.2 to 3.2) after 10 minutes of treatment. Since the test substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage (Westernik, 2017).

Justification for classification or non-classification

Skin irritation:

Based on the results of an in vivo skin irritation study, the test substance is classified as Skin Irrit. 2 – H315 (Causes skin irritation) according to EU CLP (EC 1272/2008) criteria.

Eye irritation:

The test substance induced an IVIS ≤ 3 in anin vitroBovine Corneal Opacity and Permeability study and was therefore not irritating under the conditions of this test. However, given its skin irritation properties and in line with classification for substances with similar structures, a conservative classification as Eye Irrit. 2 - H319 (Causes serious eye irritation) is proposed.