Reaction products of 3-methyl-5-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylenedecahydro-1-naphthalenyl]-1-penten-3-ol, cyclized

Endpoint summary

Description of key information

Skin irritation: Irritating (GHS Classification Category 2),in vitro(RHE EpiSkin), OECD 439, 2016.

Skin corrosion: Non-corrosive, in vitro (RHE EpiDerm), OECD 431, 2017.

Eye irritation: Non-irritating, in vitro (EpiOcular), OECD 492, 2016.

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:

19 - 23 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test"

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 (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): Lot no.: 24364 kits M and L
- Production date: 21 September 2016
- Shipping date: Not reported
- Delivery date: Not reported
- Date of initiation of testing: Within 24 h of recieving kit

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out at RH: 80 - 100% (actual range 53 - 90%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.6 - 37.3°C).
- Temperature of post-treatment incubation (if applicable): See above.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Volume and number of steps not reported. PBS was used as a washng solution.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM).
- Incubation time: 3 h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: Not reported
- Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: Not reported

NUMBER OF REPLICATE TISSUES:

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE (N/A as MTT interference was discounted through experimentation)
- N/A
- Procedure used to prepare the killed tissues (if applicable): N/A
- N. of replicates : N/A
- Method of calculation used: N/A

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin 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 item considered non-corrosive (viability 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
- The test substance is considered to be non-corrosive to skin 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%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: N/A

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): Undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): N/A

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8 N

Duration of treatment / exposure:

3 minutes (2 tissues) and 60 mins (2 tissues)

Duration of post-treatment incubation (if applicable):

Incubated in MTT-medium for 3 hours followed by a formazan extraction period overnight.

Number of replicates:

2 per test group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure

Value:

104

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of corrosion

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure

Value:

89

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of corrosion

Table 1:       Mean adsorption (OD₅₇₀) in thein vitroskin corrosion test with the test item

	3-minute exposure			60-minute exposure
	Rep A	Rep B	Mean ± SD	Rep A	Rep B	Mean ± SD
Negative control	1.526	1.586	1.556 ± 0.042	1.770	1.588	1.679 ± 0.129
Test item	1.581	1.654	1.618 ± 0.052	1.700	1.277	1.488 ± 0.299
Positive control	0.180	0.155	0.167 ± 0.018	0.205	0.163	0.184 ± 0.030

 

Table 2:       Mean tissue viability in the in vitro skin corrosion test with the test item (%)

	3-minute exposure viability (percentage of control)	60-minute exposure viability (percentage of control)
Negative control	100	100
Test item	104	89
Positive control	11	11

 

Table 3:       Coefficient of variance between tissue replicates (%)

	3-minute exposure	60-minute exposure
Negative control	3.8	10
Test item	4.4	25
Positive control	14	21

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this summary.

Executive summary:

The skin corrosion potential of the test item was evaluated in accordance with the OECD 431 guidance document and GLP using the reconstructed human epidermis (RHE) skin model.

The test item was tested through topical application for 3 minutes and 1 hour on a human three dimensional epidermal model (EpiDerm (EPI-200)). The test item was heated up to 60°C and applied undiluted (50 µl) directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 11% 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 ≤ 25%, indicating that the test system functioned properly.

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 89%, 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.

It is concluded that this test is valid and that the test item is not corrosive in thein vitro skin corrosion test under the experimental conditions described in this summary.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 - 10 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small model (EPISKIN-SM)
- Tissue batch number(s): 16-EKIN-040
- Production date: 04 October 2016
- Shipping date: Not reported
- Delivery date: Not reported
- Date of initiation of testing: 04 October 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35.7 - 37.2 ºC
- Temperature of post-treatment incubation (if applicable): 37 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Not reported bu tissues were washed.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: N/A

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in PBS
- Incubation time: 3 h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: Not reported
- Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Not reported
- Barrier function: Not reported
- Morphology: Not reported
- Contamination: Not reported
- Reproducibility: Not reported

NUMBER OF REPLICATE TISSUES: 3 per test group

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: Not required
- Procedure used to prepare the killed tissues (if applicable): N/A
- N. of replicates : N/A
- Method of calculation used: N/A

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

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

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): N/A

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5 %

Duration of treatment / exposure:

15 mins

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3 per test group

Irritation / corrosion parameter:

% tissue viability

Value:

23

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: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

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: Yes

Table 1: Mean absorption in thein vitroskin irritation test

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	1.205	1.154	1.110	1.156	±	0.047
The test item	0.339	0.243	0.212	0.265	±	0.066
Positive control	0.238	0.133	0.042	0.138	±	0.098

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

 

Table 2: Mean tissue viability in thein vitroskin irritation test

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	4
The test item	23	6
Positive control	12	8

 

 

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test substance was found to be irritant in the in vitro test and should be classified as Category 2 according to GHS criteria.

Executive summary:

The skin irritation potential of Reaction products of the test item using a human skin model in accordance with OECD Guideline 439 and GLP.

The test substance was heated and applied undiluted (10 µl) directly on top of the skin tissue for 15 minutes. After a 42 h post-incubation period, determination of cytotoxicity (irritancy) was performed. The mean tissue viability obtained after 15 minutes exposure to the test item was 23 %, indicating an irritant response. Under GHS classification, the test item should be classifed as Category 2 (irritant).

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:

09 - 12 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with international guidelines and in accordance with 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability

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

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live

The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

Duration of treatment / exposure:

30 ± 2 mins

Duration of post- treatment incubation (in vitro):

120 ± 15 minutes at 37 ºC

Number of animals or in vitro replicates:

2 tissues per test group

Details on study design:

EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 23438 kit A) (MatTek Corporation, Ashland MA, U.S.A)

The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

On the day of receipt, the 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 Assay Medium, which was refreshed after approximately 1 hour. Assay Medium was supplied by MatTek Corporation, Ashland, USA.

All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 59 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.1 - 37.2°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 laboratory historical data these deviations are considered not to affect the study integrity.

The test item was checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the tissues during the exposure. To assess the colour interference, 50 µl of the test item or 50 µl sterile Milli-Q water as a negative control was added to 1.0 ml Milli-Q water. The mixture was incubated for at least 1 hour at 37.0 ± 1.0°C in the dark. Furthermore, 50 µl of the test item or 50 µl sterile Milli-Q water as a negative control was added to 2.0 ml isopropanol. The mixture was incubated for 2 - 3 hours at room temperature with gentle shaking. At the end of the exposure time, the absorbance of the solutions was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. If after subtraction of the negative control, the OD for the test item solution is >0.08, the test item is considered as possibly interacting with the MTT measurement.

The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 µl of the test item was added to 1 ml MTT 1 mg/ml MTT solution. The mixture was incubated for approximately 3 hours at
37.0 ± 1.0°C in the dark. A negative control, 50 µl sterile Milli-Q water was tested concurrently. If the MTT solution colour turned blue / purple or if a blue / purple precipitate was observed the test item interacts with MTT. Only test items which bind to the tissue after rinsing can interact with MTT in the main assay.

The test was performed on a total of 2 tissues per test item together with a negative control and positive control. Before the assay was started the entire tissues was pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes.

Two tissues were treated with 50 µl MilliQ water (negative control) and 2 tissues with 50 µl Methyl Acetate (positive control) respectively. 50 µl of the undiluted test item was added into the 6-well plates on top of the tissues. After the exposure period with the test item (30 ± 2 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 12 ± 2 minutes 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 120 ± 15 minutes at 37°C.

After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 ml MTT-medium
(1.0 mg/ml). The tissues were incubated for 180 ± 10 minutes at 37°C. After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labelled 24-well plate containing 2.0 ml of isopropanol in each designated well so that isopropanol is flowing into the insert on the tissue surface. Formazan was extracted with 2 ml isopropanol refrigerated for 18 ± 2 hours in the dark. At the end of the extraction period, the liquid within each insert was decanted/pipetted into the well from which it was taken. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

A test item is considered to be positive in the in vitro eye irritation test if:

The relative mean tissue viability of two individual tissues after exposure to the test item is ≤ 60% of the mean viability of the negative controls, requiring classification for eye irritation or serious eye damage (UN GHS Category 1 or 2).

A test item is considered to be negative in the in vitro eye irritation test if:

The relative mean tissue viability of two individual tissues after exposure to the test item is > 60% of the mean viability of the negative controls, requiring no classification for eye irritation or serious eye damage (UN GHS No Category).

Irritation parameter:

other: cell viability (%)

Run / experiment:

Undiluted test item

Value:

97

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.
- Acceptance criteria met for positive control: The mean relative tissue viability of the positive control should be <50% relative to the negative control.
- Range of historical values if different from the ones specified in the test guideline: N/A

Table 1: Mean adsorption values

	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.713	1.842	1.778	±	0.091
Test item	1.509	1.927	1.718	±	0.296
Positive control	0.589	0.588	0.589	±	0.001

OD = optical density

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.041). Isopropanol was used to measure the background absorption.

 

Table 2: Mean tissue viability

	Mean tissue viability (percentage of control)	Standard deviation  (percentage)
Negative control	100	5.1
Test item	97	17
Positive control	33	0.03

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is non-irritant in the EpiOcular™ test under the experimental conditions previously described.

Executive summary:

After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

The positive control had a mean cell viability of 33% after 30±2 minutes exposure. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues was within 0.5 and 2.8. The standard deviation value of the percentage viability of two tissues treated identically was less than 17%, indicating that the test system functioned properly.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30±2 minutes treatment with the test item compared to the negative control tissues was 97%. Since the mean relative tissue viability for the test item was above 60% after 30 ± 2 minutes treatment the test item is considered to be non-irritant.

Finally, it is concluded that this test is valid and that the test item is non-irritant in the EpiOcular™ test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

OECD 439, 2016 - The skin irritation potential of the test item using a human skin model in accordance with OECD Guideline 439 and GLP. The test substance was heated (to liquefy) and applied undiluted (10 µL) directly on top of the skin tissue for 15 minutes. After a 42 -h post incubation period, determination of cytotoxicity (irritancy) was performed. The mean tissue viability obtained after 15 minutes exposure to the test item was 23 %, indicating an irritant response. Under GHS classification, the test item should be classified as Category 2 (irritant).

 

OECD 431, 2017 - The skin corrosion potential of the test item was evaluated in accordance with the OECD 431 guidance document and GLP using the reconstructed human epidermis (RHE) skin model. The test item was tested through topical application for 3 minutes and 60 minutes on a human three-dimensional epidermal model (EpiDerm (EPI-200)). The test item was heated up to 60°C and applied undiluted (50 µl) directly on top of the skin tissue. The positive control had a mean relative tissue viability of 11% 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 ≤ 25%,indicating that the test system functioned properly. 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 89%, 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. It is concluded that this test was valid and that the test item is not corrosive in thein vitroskin corrosion test.

 

OECD 492, 2016 - After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The positive control had a mean cell viability of 33% after 30±2 minutes exposure. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues was within 0.5 and 2.8. The standard deviation value of the percentage viability of two tissues treated identically was less than 17%, indicating that the test system functioned properly. Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30±2 minutes treatment with the test item compared to the negative control tissues was 97%. Since the mean relative tissue viability for the test item was above 60% after 30 ± 2 minutes treatment the test item is considered to be non-irritant.

 

Justification for classification or non-classification

Skin irritation - The test item meets the classification criteria under Regulation (EC) No 1272/2008 for skin irritancy (category 2).

Skin corrosion - The test item does not meet the classification criteria under Regulation (EC) No 1272/2008 for skin corrosion.

Eye irritation - The test item does not meet the classification criteria under Regulation (EC) No 1272/2008 for eye irritation.