Phenol, C14-18-alkyl derivs.

Endpoint summary

Description of key information

SKIN CORROSION/IRRITATION
Not corrosive (in vitro); OECD 431 and EU Method B.40 BIS
Not irritating (in vitro); OECD 439 and EU Method B.46
EYE IRRITATION
Not irritating (in vitro); OECD 437 and EU Method B.47
Not irritating (in vivo); OECD 405, EU Method B.5, EPA OPPTS 870.2400 and JMAFF, 12 Nousan, Notification No 8147

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 August 2014 to 29 August 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test"

Deviations:

no

GLP compliance:

yes

Species:

other: EpiDerm Skin Model

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

TEST SYSTEM
EpiDerm Skin Model (EPI-200, Lot no.:20565 Lot R, MatTek Corporation, Ashland MA, USA).
The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Type of coverage:

open

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: tissues were exposed to both positive (8 N potassium hydroxide) and negative (Milli-Q water) concurrent controls

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL. The liquid test material was applied undiluted directly on top of the tissue.

Duration of treatment / exposure:

3 minutes and 1 hour

Observation period:

After exposure the skin tissue was thoroughly rinsed to remove the test material, followed by determination of the cytotoxic (corrosive) effect.

Number of animals:

Two tissue samples were exposed for 3 minutes and two were exposed for 1 hour.

Details on study design:

CELL CULTURE
- Tissues: On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL Dulbecco’s Modified Eagle’s Medium (supplemented DMEM medium, serum-free).
- MTT medium: MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM).
- Environmental conditions: All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 to 100 % (actual range 68 to 84 %), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.3 to 36.6 °C).

TEST FOR REDUCTION OF MTT BY THE TEST MATERIAL
The test material was checked for possible direct MTT reduction before the study was started. To assess the ability of the test material to reduce MTT, 100 µL of the test material was added to 1 mL MTT medium. The mixture was incubated for approximately 1 hour at room temperature in the dark. A negative control, sterile Milli-Q water was tested concurrently.

APPLICATION/TREATMENT OF THE TEST MATERIAL
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM medium per well. The level of the DMEM medium was just beneath the tissue. The plates were incubated for 3 hours at 37.0 ± 1.0 °C. The medium was replaced with fresh DMEM medium just before the test material was applied. The undiluted test material was added with a pipette.
After the exposure period, the tissues were washed with phosphate buffered saline (PBS) to remove residual test material. Rinsed tissues were kept in 24 well plates on 300 µL DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

CELL VIABILITY MEASUREMENT
The DMEM medium was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at 37 °C in air containing 5 % CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol over night at room temperature.
The amount of extracted formazan was determined spectrophotometrically at 540 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues.
Skin corrosion potential of the test material was classified according to remaining cell viability following exposure of the test material with either of the two exposure times.

Irritation / corrosion parameter:

other: other: Relative mean tissue viability

Value:

92

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 3 minute exposure. Max. score: 100.0. Reversibility: no data. Remarks: The value was 92 %. (migrated information)

Irritation / corrosion parameter:

other: other: Relative mean tissue viability

Value:

89

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 1 hour exposure. Max. score: 100.0. Reversibility: no data. Remarks: The value was 89 %. (migrated information)

Irritant / corrosive response data:

The test material was checked for possible direct MTT reduction by adding it to MTT medium. As no colour change was observed, it was concluded that the test material did not interact with MTT.

The mean absorption at 540 nm measured after treatment with the test material and controls are presented in Table 1. Table 2 shows the mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissues.
Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 92 % and 89 %, respectively. As the mean relative tissue viability was not below 50 % after 3 minutes of treatment and not below 15 % after 1 hour of treatment, the test material is considered to be not corrosive.
The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 8 %. The maximum inter-tissue variability in viability between two tissues treated identically was less than 4 % and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 2 %. It was therefore concluded that the test system functioned properly.

Table 1: Mean absorption in the in vitro skin corrosion test

	3- Minute Application			1-Hour Application
	A (OD540)	B (OD540)	Mean ± SD	A (OD540)	B (OD540)	Mean ± SD
Negative Control	1.823	1.768	1.796 ± 0.039	1.858	1.859	1.859 ± 0.001
Test Material	1.681	1.618	1.650 ± 0.044	1.656	1.665	1.660 ± 0.006
Positive Control	0.145	0.146	0.146 ± 0.001	0.130	0.127	0.128 ± 0.002

SD = Standard deviation

Duplicate exposures are represented by A and B

Values are corrected for background absorption (measured using isopropanol)

 

Table 2: Mean tissue viability in the in vitro skin corrosion test

	3- Minute Application Viability (% of control)	1-Hour Application Viability (% of control)
Negative Control	100	100
Test Material	92	89
Positive Control	8	7

Interpretation of results:

other: Not corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.

Executive summary:

The corrosion potential of the test material was investigated in an in vitro skin corrosion test using a human skin model in accordance with the standardised guidelines OECD 431 and EU Method B.40 BIS under GLP conditions.

The study determined the ability of the test material to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential was tested through topical application for 3 minutes and 1 hour. The test material was applied undiluted (50 µL) directly on top of the skin tissue. Tissues were also exposed to both positive (8 N potassium hydroxide) and negative (Milli-Q water) concurrent controls.

Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 92 % and 89 %, respectively. As the mean relative tissue viability for the test material was not below 50 % after the 3-minute treatment and not below 15 % after the 1-hour treatment, it is considered to be not corrosive.

The positive control had a mean relative tissue viability of 8 % after 3 minutes of exposure. The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The maximum inter-tissue variability in viability between two tissues treated identically was less than 4 % and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 2 %, indicating that the test system functioned properly. It was concluded that the test was valid.

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.

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

Study period:

08 September 2014 to 22 September 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF, 12 Nousan, Notification No 8147

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Strain: Albino rabbit, New Zealand White, (SPF-Quality)
- Age at study initiation: 12 to 24 weeks
- Weight at study initiation: At least 1.5 kg
- Housing: Animals were individually housed in cages with perforated floors (67 x 62 x 55 cm) and shelters (40 x 32 x 23 cm).
- Diet: Pelleted diet for rabbits, approximately 100 grams per day; hay and wooden sticks
- Water: ad libitum access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24 °C
- Humidity: 40 to 70 % (relative)
- Air changes: At least 10/hour
- Photoperiod: 12-hour light/12-hour dark cycle

Vehicle:

unchanged (no vehicle)

Controls:

other: The other eye remained untreated and served as the reference control.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.1 mL

Duration of treatment / exposure:

Following installation of the test material, the lids were gently held together for about 1 second to prevent loss of the test material.

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

3 females

Details on study design:

PRE-EMPTIVE PAIN MANAGEMENT
One hour prior to instillation of the test material, buprenorphine (Buprenodale®, Dechra Ltd., Stoke-on-Trent, United Kingdom) 0.01 mg/kg was administered by subcutaneous injection in order to provide a therapeutic level of systemic analgesia.
Five minutes prior to instillation of the test material, two drops of the topical anaesthetic alcaine 0.5 % (SA Alcon-Couvreur NV, Puurs, Belgium) were applied to both eyes.

SCORING SYSTEM
The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7 days after instillation of the test material. The irritation scores and a description of all other (local) effects were recorded.
The irritation was assessed according to the numerical scoring system given in Table 1. At each observation, the highest scores given were recorded.

TOOLS USED TO ASSESS SCORE
Where standard lighting was considered inadequate for observing minor effects, eye examinations were performed using an ophthalmic examination lamp. Immediately after the 24-hour observation, a solution of 2 % fluorescein in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage.
Immediately after fluorescein examination on Day 2, in order to provide a continued level of systemic analgesia, buprenorphine 0.01 mg/kg and meloxicam (Metacam®, Boehringer Vetmed GmbH, Ingelheim/Rhein, Germany) 0.5 mg/kg were administered by subcutaneous injection.

OTHER OBSERVATIONS
- Mortality/Viability: Twice daily
- Toxicity At least once daily
- Body Weight: Day of treatment (prior to instillation) and after the final observation.
- Sacrifice: After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20 % (AST Farma BV, Oudewater, The Netherlands). No necropsy was performed.

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

7 d

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7d

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

48 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7d

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7d

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

other: 1h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7d

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

7 d

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7d

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

48 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7d

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7d

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

other: 1h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7d

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

7 d

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

48 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 d

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7d

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

other: 1h

Score:

1

Max. score:

1

Reversibility:

not specified

Irritation parameter:

iris score

Basis:

animal #3

Time point:

7 d

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

iris score

Basis:

animal #3

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

iris score

Basis:

animal #3

Time point:

48 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

iris score

Basis:

animal #3

Time point:

other: 1h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

iris score

Basis:

animal #2

Time point:

7 d

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

iris score

Basis:

animal #2

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

iris score

Basis:

animal #2

Time point:

48 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

iris score

Basis:

animal #2

Time point:

other: 1h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

iris score

Basis:

animal #1

Time point:

7 d

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

iris score

Basis:

animal #1

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

iris score

Basis:

animal #1

Time point:

48 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

iris score

Basis:

animal #1

Time point:

other: 1h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

7 d

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

48 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

other: 1h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

7 d

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

48 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

other: 1h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

7 d

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

48 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

other: 1h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

7 d

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

48 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 48h

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

other: 1hr

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 48h

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

7 d

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

48 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 48h

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

other: 1hr

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 48h

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

other: 1hr

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24 h

Score:

0

Max. score:

0

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

48 h

Score:

0

Max. score:

0

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

72 h

Score:

0

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

7 d

Score:

0

Max. score:

0

Reversibility:

fully reversible

Irritant / corrosive response data:

Instillation of the test material resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge. The irritation had completely resolved within 7 days in all animals.
No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2 % fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage.
There was no evidence of ocular corrosion.

Other effects:

No staining of (peri) ocular tissues by the test material was observed. Remnants of the test material were present on the outside of the eyelids of all animals on Days 1, 2 and/or 3.
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Table 1: Individual Eye Irritation Scores

Animal	Time After Dosing	Corneal Opacity	Iris	Conjunctivae
				Redness	Chemosis	Discharge
785	1 hour	0	0	1	1	0
	24 hours	0	0	2	0	1
	8 hours	0	0	1	0	0
	72 hours	0	0	1	0	0
	7 days	0	0	0	0	0
786	1 hour	0	0	1	1	1
	24 hours	0	0	2	1	1
	8 hours	0	0	1	0	0
	72 hours	0	0	1	0	0
	7 days	0	0	0	0	0
787	1 hour	0	0	1	1	1
	24 hours	0	0	2	1	1
	8 hours	0	0	1	0	0
	72 hours	0	0	1	0	0
	7 days	0	0	0	0	0

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test material was found to be non-irritating to the eye.

Executive summary:

The potential of the test material to cause acute eye irritation or corrosion was investigated in a study conducted in accordance with the standardised guidelines OECD 405, EU Method B.5, EPA OPPTS 870.2400 and JMAFF, 12 Nousan, Notification No 8147 under GLP conditions.

Single samples of 0.1 mL of the test material were instilled into one eye of each of three New Zealand White rabbits. The other eye remained untreated and served as the reference control. Observations were made 1, 24, 48 and 72 hours and 7 days after instillation.

Instillation of the test material resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge. The irritation had completely resolved within 7 days in all animals. No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2 % fluorescein 24 hours after test material instillation revealed no corneal epithelial damage.

Remnants of the test material were present on the outside of the eyelids of all animals on Days 1, 2 and/or 3.

Under the conditions of this study, the test material was found to be non-irritating to the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion/Irritation

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

The study determined the ability of the test material to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential was tested through topical application for 3 minutes and 1 hour. The test material was applied undiluted (50 µL) directly on top of the skin tissue. Tissues were also exposed to both positive (8 N potassium hydroxide) and negative (Milli-Q water) concurrent controls.

Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 92 % and 89 %, respectively. As the mean relative tissue viability for the test material was not below 50 % after the 3-minute treatment and not below 15 % after the 1-hour treatment, it is considered to be not corrosive.

The positive control had a mean relative tissue viability of 8 % after 3 minutes of exposure. The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The maximum inter-tissue variability in viability between two tissues treated identically was less than 4 % and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 2 %, indicating that the test system functioned properly. It was concluded that the test was valid.

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.

 

The irritation potential of the test material was investigated in an in vitro skin irritation test using a human skin model in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The study determined the ability of the test material to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SM™)). The possible skin irritation potential of the test material was tested through topical application for 15 minutes. The test material was applied undiluted (25 µL) directly on top of the skin tissue for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Tissues were also exposed to both positive (5 % aqueous sodium dodecyl sulphate in phosphate buffered saline) and negative (phosphate buffered saline) concurrent controls.

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 the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 15 minutes of treatment compared to the negative control tissues was 118 %. Since the mean relative tissue viability for the test material was above 50 % after 15 minutes of treatment, the test material is considered to be non-irritant.

The positive control had a mean cell viability of 13 % after 15 minutes of 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 12 %, indicating that the test system functioned properly. It was concluded that the test was valid.

Under the conditions of this study, the test material is a non- irritant in the in vitro skin irritation test.

 

Eye Irritation

Screening for the eye irritancy potential of the test material in vitro was carried out using the Bovine Corneal Opacity and Permeability test (BCOP test). The study was conducted in accordance with the standardised guidelines OECD 437 and EU Method B.47 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Bovine eyes were used as soon as possible after slaughter on the same day. The possible ocular irritancy was tested through topical application of the undiluted test material (750 µL) for 10 ± 1 minutes directly on top of the corneas. Following the exposure period, the corneas were assessed for opacity and permeability. Additional corneas were exposed to both positive (10 % benzalkonium chloride in physiological saline) and negative (physiological saline) concurrent controls.

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 was 125 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The test material did not induce ocular irritation in any cornea for both endpoints (opacity and permeability), resulting in a mean in vitro irritancy score of 2.1 after 10 minutes of treatment.

It was concluded that the test is valid and that the under the conditions of this study, the test material is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test.

Since the test material induced an IVIS = 3, no classification is required for eye irritation or serious eye damage.

 

The potential of the test material to cause acute eye irritation or corrosion in vivo was investigated in a study conducted in accordance with the standardised guidelines OECD 405, EU Method B.5, EPA OPPTS 870.2400 and JMAFF, 12 Nousan, Notification No 8147 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Single samples of 0.1 mL of the test material were instilled into one eye of each of three New Zealand White rabbits. The other eye remained untreated and served as the reference control. Observations were made 1, 24, 48 and 72 hours and 7 days after instillation.

Instillation of the test material resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge. The irritation had completely resolved within 7 days in all animals. No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2 % fluorescein 24 hours after test material instillation revealed no corneal epithelial damage.

Remnants of the test material were present on the outside of the eyelids of all animals on Days 1, 2 and/or 3.

Under the conditions of this study, the test material was found to be non-irritating to the eye.

Justification for selection of skin irritation / corrosion endpoint:
This endpoint was addressed using two in vitro studies; one assessed the test material for skin corrosion potential and the other assessed the test material for irritation potential. Therefore the in vitro corrosion study was selected as key.

Justification for selection of eye irritation endpoint:
This endpoint was addressed using one in vitro study and one in vivo study; both screened the test material for eye irritation potential. The results were complementary; therefore the in vivo study was selected as key.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to skin and eye irritation or corrosion.