2-o-tolylethanol

Endpoint summary

Description of key information

Skin corrosion: Substance is not skin irritating and therefore also not corrosive

Skin irritation (OECD TG 439): Not skin irritating

Eye irritation (OECD TG 438): Serious eye damage

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

Study period:

14 March 2016 - 21 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

in accordance with GLP conditions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2015)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2012)

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France.

Source strain:

other: Not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM
- Tissue batch number: 16-EKIN-011
- Production / shipping / delivery date: No data
- Date of initiation of testing: 14 March 2016

PRE-TEST PROCEDURE:
- Pre-incubation: On day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for approximately 22 hours at 37°C.
- Test item colour interference: To assess colour interference, 10 μL of Peomosa (mono-constituent) was added to 90 μL Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μL Milli-Q water was tested concurrently. At the end of the shaking period a colour check was performed.
- Test item MTT reduction: To assess the ability of the test item to reduce MTT, 25 μL of the test item was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently. At the end of the incubation period a colour check was performed.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure / post-treatment incubation: 36.3 - 37.3 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
Tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml in PBS
- Incubation time: 3 hours at 37 °C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: No data
- Linear OD range of spectrophotometer: No data

DECISION CRITERIA
- 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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Test material
- Applied volume: 25 μL

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Relative mean tissue viability compared to the negative control tissues (100%)

Value:

74

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:
- Direct-MTT reduction: No colour changes observed
- Colour interference with MTT: Not colour changes observed

DEMONSTRATION OF TECHNICAL PROFICIENCY: No data

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 44%. The positive control meets the validity criterion meets the validity criterion even though it is just outside the historical control range, which has not affected the result of the results for the test substance.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was less than 17%, indicating that the test system functioned properly.

Mean OD570 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item	OD570 of tissues	Mean OD562 of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)
Negative Control Item	1.066	1.047	0.022		100
	1.052
	1.024
Positive Control Item	0.398	0.462	0.078	37	44
	0.549			52
	0.437			43
Test Item	0.796	0.770	0.163	75	74
	0.595			57
	0.919			90

SD = Standard deviation

*The mean viability of the negative control tissues is set at 100%.

Interpretation of results:

other: not skin irritating

Remarks:

According to EU CLP 1272/2008 and its amendments.

Conclusions:

Under the conditons of this test, the relative mean tissue viability for the test item determined to be 74%. Based on this result, the substance is considered to be non-irritant and does not need to be classified for skin irritation in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC) and its amendments.

Executive summary:

The possible skin irritation potential of Peomosa was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 µL undiluted test substance. After 42 hours 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 (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 44% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 74%. Based on this result, the substance is considered to be non-irritant and does not need to be classified for skin irritation in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC)

and its amendments

.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

22 March 2016 - 4 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

in accordance with GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

other: ROSS, spring chickens

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Number of animals: No data
- Characteristics of donor animals: male/female, approximately 7 weeks of age and 1.5-2.5 kg
- Storage, temperature and transport conditions of ocular tissue: Transport in small plastic boxes on tissues moistened with isotonic saline at ambient temperature
- Time interval prior to initiating testing: Within 2 hours after kill
- Indication of any existing defects or lesions in ocular tissue samples: Only undamaged eyes used
- Indication of any antibiotics used: No data

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

240 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
- Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
- The preparation and validation of the eyes prior to the ICE-test were all according to OECD guideline 438.

EQUILIBRATION AND BASELINE RECORDINGS
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. After an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured to determine the zero reference value for corneal swelling calculations.

OBSERVATION PERIOD
Examination of the eyes was performed after 0, 30, 75, 120, 180 and 240 minutes

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The eyes were rinsed with 20 mL saline after 10 seconds exposure

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity, fluorescein retention and swelling: slit-lamp microscope (Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland)
- Macroscopic morphological damage to the surface: microscope
- Others: histopathology (preserved with 4% formaldehyde and stained with PAS)

SCORING SYSTEM:
- Mean corneal swelling (%), mean maximum opacity score and mean fluorescein retention score at 30 minutes post-treatment: According to criteria specified in OECD TG 438

DECISION CRITERIA: According to OECD TG 438.

Irritation parameter:

percent corneal swelling

Run / experiment:

Slit-lamp examination

Value:

13

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Maximum mean score

Irritation parameter:

cornea opacity score

Run / experiment:

Slit-lamp examination

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Maximum mean score

Irritation parameter:

fluorescein retention score

Run / experiment:

Slit-lamp examination

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Microscopic examination of the corneas treated with Peomosa revealed very slight or slight erosion, moderate necrosis (two corneas) and moderate and severe vacuolation (two corneas) of the epithelium. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight, moderate or severe erosion and very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane (one cornea), and endothelial necrosis (one cornea).

DEMONSTRATION OF TECHNICAL PROFICIENCY: This OECD TG was developed at this CRO and therefore the test has been performed for many years.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate.
- Acceptance criteria met for positive control: The positive control BAC 5% caused moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Interpretation of results:

other: Category 1 (serious eye damage)

Remarks:

According to EU CLP 1272/2008 and its amendments.

Conclusions:

Under the test conditions (OECD 438 and GLP), the test substance is considered to cause serious damage to the eye and should be classified as such (Eye Dam. 1 / H318) in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC) and its amendments.

Executive summary:

In accordance with OECD guideline 438 and GLP, the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) Test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. After the exposure the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. The test substance caused corneal effects consisting of slight corneal swelling (mean of 13%), severe opacity (mean score of 3.0) and severe fluorescein retention (mean score of 3.0). In addition, loosening of epithelium was observed. Microscopic examination of the corneas revealed very slight or slight erosion, moderate necrosis (two corneas) and moderate and severe vacuolation (two corneas) of the epithelium. The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. Microscopic examination did not reveal any abnormalities. The positive control BAC 5% caused moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination revealed slight, moderate or severe erosion and very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane (one cornea), and endothelial necrosis (one cornea). Based on these results, the test substance is considered to cause serious damage to the eye and should be classified as such (Eye Dam. 1 / H318) in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC)

and its amendments

.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Additional information

In vitro skin irritation

The skin irritation potential of Peomosa was tested in vitro using a human skin model in accordance with OECD TG 439 under GLP conditions. Reliable negative and positive controls were included. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was found to be 74%. Based on this result, the substance is considered to be non-irritant.

In vitro eye irritation

The eye irritating potential of Peomosa was tested in an Isolated Chicken Eye (ICE) Test in accordance with OECD TG 438 and under GLP conditions. The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. The test substance caused corneal effects consisting of slight corneal swelling (mean of 13%), severe opacity (mean score of 3.0) and severe fluorescein retention (mean score of 3.0). In addition, loosening of epithelium was observed. Microscopic examination of the corneas revealed very slight or slight erosion, moderate necrosis (two corneas) and moderate and severe vacuolation (two corneas) of the epithelium. Based on these results, the test substance is considered to cause serious damage to the eye.

Justification for classification or non-classification

Based on the negative result found in the in vitro skin irritation test, the substance does not need to be classified as a skin irritant in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC) and its amendments.

Based on the positive result found in the ICE test the substance is considered to cause serious eye damage. In accordance the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC) and its amendments, this results in a Category 1 classification for this endpoint (H318: Causes serious eye damage).