Registration Dossier

Administrative data

Description of key information

- Skin irritation (OECD 439): not irritating

- Eye irritation (OECD 405): irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
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:
adopted 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:
2008, amended 20 July 2012
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EPISKIN Small Model TM
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-SM TM, 0.38 cm² (SkinEthic Laboratories, Lyon, France)
- Tissue batch number(s): 19-EKIN-024
- Date of initiation of testing: 11 Jun 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.
- Observable damage in the tissue due to washing: Not reported.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h (at 37°C)
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: IC50 = 2.1 mg/mL (1.5 mg/mL≤ IC50 ≤ 3.0 mg/mL
- Morphology: HES stained paraffin section: Multi-layered, highly differentiated epidermis consisting of organized basal, spinous and granular layers, and a multilayered stratum corneum (result is satisfactory) / number of cell layers ≥ 4
- Contamination: On blood of the donors the absence of HIV1 and 2 antibodies, hepatitis C antibodies and hepatitis B antigen HBs has been verified; on cells from donors, the absence of bacteria, fungus and mycopasma has been varified.

NUMBER OF REPLICATE TISSUES: triplicate

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

PREDICTION MODEL / DECISION CRITERIA :
A test item 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 item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item 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 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): 25 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL
- Concentration (if solution): 5%
The positive control was re-spread after 7 minutes contact time.
Duration of treatment / exposure:
15 ± 0.5 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
triplicate for each treatment and control group
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 3 tissues
Run / experiment:
15 min exposure
Value:
96
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: Not reported.
- Direct-MTT reduction: Not reported.
- Colour interference with MTT: Because no color changes were observed it was concluded that the test item did not interact with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues (1.376 ± 0.056) was within the laboratory historical control data range (0.422 - 1.547; mean: 0.96, SD: 0.17, n = 174).
- Acceptance criteria met for positive control: The absolute mean OD570 of the positive control tissues (0.123 ± 0.020) was within the laboratory historical control data range (0.023 - 0.449; mean: 0.12, SD: 0.09, n = 173).
The standard deviation value of the percentage viability of three tissues treated identically was < 5% (negative control 4.1%, positive control 1.5% and test item 3.9%), indicating that the test system functioned properly.

Table 1: MTT assay after 15 min exposure

  Negative control Positive control Test item
Tissue sample 1 2 3 1 2 3 1 2 3
OD570 1.388 1.426 1.315 0.107 0.146 0.115 1.258 1.346 1.355
OD570(mean of 3 tissues) 1.376 ± 0.056 0.123 ± 0.020 1.320 ± 0.053
Viability (%) 100 8.9 96
SD 4.1 1.5 3.9
Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
GLP compliance:
yes (incl. QA statement)
Remarks:
Food and Consumer Product Safety Authority (VWA), Den Haag, The Netherlands
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Harlan, France
- Age at study initiation: at least 6 weeks
- Weight at study initiation: at least 1.0 kg
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 21.4
- Humidity (%): 42 - 70
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light):12 hrs

Vehicle:
unchanged (no vehicle)
Controls:
other: one eye was untreated and served as the control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.1 mL
- Concentration: 100%

Duration of treatment / exposure:
lids held together for 1 sec
Observation period (in vivo):
24, 48, and 72 hour, 7 days, and 14 days after exposure
Number of animals or in vitro replicates:
3
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): not done

SCORING SYSTEM: Draize

TOOL USED TO ASSESS SCORE: fluorescein
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
other: iritis (score 1) observed 1 h after dosing; reversible within 24 h
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.67
Max. score:
2
Reversibility:
not fully reversible within: 72 h
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.67
Max. score:
2
Reversibility:
fully reversible within: 72 h
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.67
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
2.33
Max. score:
3
Reversibility:
fully reversible within: 14 days
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 14 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 h
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.33
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1.3
Max. score:
4
Reversibility:
fully reversible within: 7 days
Other effects:
No clinical signs of toxicity noted
Interpretation of results:
other: Eye Irritation Cat. 2 (H319) according to Regulation (EC) No 1272/2008
Conclusions:
2-PO (MPKO) is irritating to rabbit eyes.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation / corrosion

The skin irritation properties of 2-PO (CAS 623-40-5) were tested in an in vitro skin irritation study according to OECD 439 and in compliance with GLP (Key, 2019). Three tissues of the human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)) were treated for 15 minutes. The test item was applied undiluted (25 µL), directly on top of the skin tissue. After a 42 hour 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 item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 96%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant. The positive control had a mean cell viability of 8.9% 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 < 5%, indicating that the test system functioned properly. In conclusion, 2-PO is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Eye irritation

In an in vivo eye irritation study with2-PO (CAS 623-40-5)performed under GLP according to OECD Guideline 405 0.1 mL test substance was instilled into the eye of each of 3 New Zealand White rabbits without washing (Key, 2007). The respective untreated eye served as control. The eyes were examined and graded for ocular reaction at 24, 48, 72 h, 7 and 14 days following instillation of test material into the eyes. The instillation of the test material resulted in mean cornea opacity scores (24/48/72 h) of 0 (animal 1) / 1 (animal 2) / 1 (animal 3), mean iris scores (24/48/72 h) of 0 (animal 1) / 0.67 (animal 2) / 0.67 (animal 3), mean chemosis scores (24/48/72 h) of 0.33 (animal 1) / 1.33 (animal 2) / 1.3 (animal 3) and mean conjunctivae redness scores (24/48/72 h) of 0.67 (animal 1) / 2.33 (animal 2) / 2 (animal 3). The observed effects were all reversible within 72 h and 14 days after exposure. Based on the results of the conducted study, the undiluted test material did show irritant potential towards the eyes.

Justification for classification or non-classification

The available data on skin irritation / corrosion do not meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.

The available data on eye irritation meet the criteria for classification according to Regulation (EC) No 1272/2008 and therefore the test substance will be classified as Eye Irrit. Cat. 2 (H319) according to Regulation (EC) 1272/2008.