(5E)-3,5-dimethyloct-5-en-4-ol

Endpoint summary

Description of key information

Skin irritation: non-irritating, OECD TG 404, 2018

Skin irritation: non-irritating (15 minute/42 hour: relative mean viability = 82.2%), OECD TG 439, 2018

Eye irritation: irritating, OECD TG 405, 2018

Eye irritation: no prediction could be made (ICE class 3 x III), OECD TG 438, 2018

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05-04-2018 to 16-04-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant 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)

Remarks:

inspected: July 2017 ; signature: November 2017

Test system:

human skin model

Remarks:

EPISKIN TM Small Model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM Small Model
- Tissue batch number(s): Lot no.: 18-EKIN-015
- Production date: Not reported.
- Shipping date: Not reported.
- Delivery date: 04-04-2018 ; day prior to test initiation (ca. 24 hours)
- Date of initiation of testing: ca. 05-04-2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C, 5% CO2
- Temperature of post-treatment incubation (if applicable): 37 °C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours. Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination. The tissues were then processed for MTT-loading and 3 hour incubation at 37 °C, 5% CO2.
- Observable damage in the tissue due to washing: Not applicable.
- Modifications to validated SOP: Not applicable.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT solution prepared in assay medium
- Incubation time: The plate was incubated (37 °C, 5% CO2) for 3 hours.
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm (OD570)
- Filter: Without reference filter
- Filter bandwidth: No reported.
- Linear OD range of spectrophotometer: Not reported.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Within NC and PC historic control ranges presented within the report.
- Barrier function: See above.
- Morphology: See above.
- Contamination: Not applicable.
- Reproducibility: The relative mean tissue viability for the positive control treated tissues was 9.1% relative to the negative control following the 15-minute exposure/42-hour incubation period. With standard deviation of 4.9%. The positive control acceptance criterion was therefore satisfied. The negative control triplicate treated tissues mean OD570 was 0.736 and the standard deviation of viability was 11.5%. The acceptance criterion was therefore satisfied. The test item triplicate treated tissues viability standard deviation was 1.5%. The mean OD570 for both the negative and positive controls were within the historical control ranges.
- Other: As an additional quality control step, the results of the assay are compared to the historical ranges for negative and positive controls generated by the testing laboratory to confirm the functioning of the test system.

NUMBER OF REPLICATE TISSUES: Three (3), triplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: In the pretest for colour interference: the solution containing the test item was colourless. It was therefore unnecessary to run colour correction tissues. In the pre-test for direct MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT. It was unnecessary to run killed tissues.
- Procedure used to prepare the killed tissues (if applicable): Not applicable.
- N. of replicates : Not applicable.
- Method of calculation used: Not applicable.
- Other: In the assessment of Colour Interference with the MTT endpoint, the solution containing the test item did not become coloured. This was taken to indicate the test item did not have the potential to cause colour interference.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One (n=1)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if e.g. the viability after 15-minutes exposure / 42-hours incubation is less than or equal to 50%,.
- The test substance is considered to be non-irritating to skin if e.g. the viability after 15-minutes exposure / 42-hours incubation is greater than 50%.
- Cut off points in accordance with OECD TG 439 and the GHS and CLP Classification systems.
- Skin irritation is expressed as the remaining cell viability after exposure to the test substance at exposure times 15-minutes / 42-hours incubation. Where necessary, direct MTT reduction, colour interference was completed.

OTHER:
EpiSkin RHE Model (Lot no.: 15-EKIN-015). The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt. to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. As a complimentary endpoint the concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-Hour post-exposure incubation period may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Pre-test checks for Direct MTT reduction and Colour Interference were completed.

Preincubation:
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert. 2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

Application of test item and rinsing:
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 μL (26.3 μL/cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.
The tissues were subsequently placed into was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination. Following 42 hour post exposure incubation the treated plates were then tested for MTT formazan extraction.

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): Not applicable.
- Concentration (if solution): Not applicable.
- Lot/batch no. (if required): Not applicable.
- Purity: Not applicable.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µl ; Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++
- Concentration (if solution): Tested as supplied (undiluted).

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µl ; The positive control item, Sodium dodecyl sulphate (SDS), was prepared as a 5% w/v aqueous solution. The positive control was formulated within 2 hours of being applied to the test system.
- Concentration (if solution): 5%

Duration of treatment / exposure:

Tissues were treated with the test item for 15 minutes and then washed with DPBS with Ca++ and Mg++ to remove residual test item.
Positive control: SDS (5% w/v) was respread after 7 minutes contact time to maintain distribution of the SDS - for the remainder of the contact period. This was not deemed necessary in the negative control or test item definitive test group.

Duration of post-treatment incubation (if applicable):

Subsequently the skin tissues were incubated for 42 hours at 37°C. Then 3 hours incubation prior to and/or MTT loading and Formazan extraction.

Number of replicates:

Triplicate (n=3) ; treatment and concurrent negative control and positive control groups

Irritation / corrosion parameter:

other: other: mean tissue viability

Value:

82.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minute exposure. Reversibility: no data. Remarks: n=3; SD = 1.5% ; Score in terms of percentage of negative control.

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Not applicable.
- Colour interference with MTT: Screened prior to test. Not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory has previously demonstrated technical proficiency. Also see historic control data.

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: Historic Control Data are presented in the full study report. All values for the control groups were within the ranges of the current Historic Control Data.

Table 1. Mean absorption and tissue viability in the in vitro skin irritation test with the test item

Item	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.826	0.736	0.085	112.2	100*	11.5
	0.657			89.3
	0.724			98.4
Positive Control Item	0.065	0.067	0.036	8.8	9.1	4.9
	0.032			4.3
	0.104			14.1
Test Item	0.606	0.605	0.011	82.3	82.2	1.5
	0.615			83.6
	0.593			80.6

OD = Optical Density

SD = Standard deviation

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

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this in vitro study, the test item is not considered to be irritating to skin.

Executive summary:

The study was performed to OECD TG 439 and EU Method B.46 under GLP to assess the skin irritation potential of the test item using a human three dimensional epidermal model (EPISKIN Small Model). Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 82.2% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 1.5%. The mean OD570 for the negative control treated tissues was 0.736 and the standard deviation value of the viability was 11.5%. The negative control acceptance criteria were therefore satisfied. The relative mean tissue viability for the positive control treated tissues was 9.1% relative to the negative control treated tissues and the standard deviation value of the viability was 4.9%. The positive control acceptance criteria were therefore satisfied. All acceptance criteria were considered to be met. Under the conditions of this study, the test item is not considered to be irritating to the skin.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-10-2018 to 15-11-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Justification for type of information:

Information as to the availability of the in vivo study is provided in 'attached justification'.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2017 ; signature: November 2017

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: recognised animal supplier
- Age at study initiation: 12 - 52 weeks
- Weight at study initiation: 3.744 or 4.074 kg
- Housing: Individually housed in suspended cages.
- Diet: certified rabbit diet ad libitum
- Water: mains drinking water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23
- Humidity (%): 30 - 70
- Air changes (per hr): at least 15
- Photoperiod: 12 hours light / 12 hours dark

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
- Concentration (if solution): Test material was used as supplied.

Duration of treatment / exposure:

4 hours

Observation period:

72 hours (initial observation); additional observations are made on Days 7 and 14 to assess the reversibility of skin reactions (as appropriate).

Number of animals:

2; following the guideline sequential testing approach

Details on study design:

TEST SITE
- Area of exposure: dorsal
- Type of wrap if used: semi-occlusive (2.5 cm x 2.5 cm cotton gauze patch secured with surgical adhesive tape)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, any residual test item removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: 4 hours

SCORING SYSTEM:
Erythema and Eschar Formation
No erythema _________________________________________________________________________0
Very slight erythema (barely perceptible) ________________________________________________1
Well-defined erythema ________________________________________________________________2
Moderate to severe erythema __________________________________________________________3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) ________________4

Oedema Formation
No oedema __________________________________________________________________________0
Very slight oedema (barely perceptible) _________________________________________________1
Slight oedema (edges of area well-defined by definite raising) _____________________________2
Moderate oedema (raised approximately 1 millimetre) ____________________________________3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) ___4
Any other skin reactions and clinical signs of toxicity, if present, were also recorded.

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 1h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: mean score (n=3)

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

1.77

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: mean score (n=3)

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 1h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: mean score (n=3)

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

1.34

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: mean score (n=3)

Irritant / corrosive response data:

Very slight erythema (score = 1) and slight edema (score = 2) were noted immediately, at 1 hour observation at treated skin sites. At the 24, 48 and 72 hours observations: very slight to well defined erythema (score = 1 or 2) and very slight to slight edema (score = 1 or 2) were observed. All effects reversed by the 7 day observation.

Other effects:

- Other adverse local effects: None reported.
- Other adverse systemic effects: One female gained bodyweight during the study (+0.0.56 g) and two females had a body weight loss (-0.004 g and - 0.010 g). Applicant assessment indicates that the bodyweight loss was very minor and there were no clinical sign correlations reported.

Table 1. Individual skin reactions

Skin Reaction	Observation Time	Individual Scores
		1 (female)	2 (female)	3 (female)
Erythema/Eschar formation	1 hour	1	1	1
	24 hours	2	2	2
	48 hours	1	2	2
	72 hours	1	2	2
	7 days	0	0	0
	Mean 24/48/72 hours	1.33	2.00	2.00
Oedema formation	1 hour	2	2	2
	24 hours	1	2	2
	48 hours	1	2	1
	72 hours	1	1	1
	7 days	0	0	0
	Mean 24/48/72 hours	1.00	1.67	1.33

Mean scores per organism at 24, 48 and 72h:

Erythemea/Escar Formation:

1: total = 4.0 ; mean score = 1.33

2: total = 6.0 ; mean score = 2.00

3: total = 6.0 ; mean score = 2.00

Edema Formation:

1: total = 3.0 ; mean score = 1.00

2. total = 5.0 ; mean score = 1.67

3: total = 4.0 ; mean score = 1.33

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the test item is not considered to be irritating.

Executive summary:

The study was performed to OECD TG 404, EU Method B.4 and the Japanese MAFF (2000) and US EPA OPPTS 870.2500 under GLP to assess the primary skin irritancy potential of the test item in accordance with GLP in New Zealand White rabbits. Following single 4-Hour, semi-occluded applications to the intact rabbit skin. 0.5 mL of the test item was introduced under a 2.5 cm x 2.5 cm cotton gauze patch and placed in position on the clipped skin to assess the irritancy potential of the test item. The patch was secured in position with a strip of surgical adhesive tape. After 4 hours of exposure to the test item, the patches were removed and individual dose sites were scored at approximately 1, 24, 48, 72 hours and 7 days, respectively. A single 4-Hour, semi occluded application of the test item to the intact skin of three rabbits produced well-defined erythema and slight edema. Treated skin sites appeared normal at the 7-Day observation. No corrosive effects were noted. No other skin reactions noted. Mean scores for following grading at 24, 48 and 72h were 1.33, 2.0 and 2.0 in erythema and eschar and 1.0, 1.67 and 1.33 in edema scoring criteria. At day 7, mean scores were zero for erythema and edema, respectively. Under the conditions of the study, the item is not considered to be a skin irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21-05-2018 to 26-09-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

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)

Remarks:

inspected: July 2017 ; signature: November 2017

Species:

other: chicken

Strain:

not specified

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): 0.03 mL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of isotonic saline.
Controls (negative and positive control items) were similarly applied to the cornea in the negative and positive control groups respectively.

Observation period (in vivo):

Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.

Number of animals or in vitro replicates:

Triplicate (n=3)

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES: Full details are provided in the full study report.

EQUILIBRATION AND BASELINE RECORDINGS: Taken at zero time after 45 minutes incubation prior to exposure.

NUMBER OF REPLICATES: Triplicate (3) for test item, positive control and negative controls.

NEGATIVE CONTROL USED: Yes,.

SOLVENT CONTROL USED (if applicable): Not applicable.

POSITIVE CONTROL USED : Benzalkonium chloride (5%)

APPLICATION DOSE AND EXPOSURE TIME: Application dose: 0.03 ml ; Exposure time: 10 seconds.

OBSERVATION PERIOD: prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after decontaminated with saline.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Test item remained in place for 10 seconds and then rinsed with 20 mL isotonic saline. Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.
- Indicate any deviation from test procedure in the Guideline: Not applicable.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Microscope.
- Damage to epithelium based on fluorescein retention: Microscope.
- Swelling: measured with optical pachymeter on a slit-lamp microscope
- Macroscopic morphological damage to the surface: Yes.
- Others (e.g, histopathology): Not applicable.

SCORING SYSTEM:
- Mean corneal swelling (%): See tables 1 to 3.
- Mean maximum opacity score: See tables 1 to 3.
- Mean fluorescein retention score at 30 minutes post-treatment: See tables 1 to 3.

DECISION CRITERIA: In accordance with guideline OECD TG 438.
Endpoints used during the evaluation procedure were corneal opacity, swelling, fluorescein retention and morphological effects (e.g. pitting, sloughing or roughening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which is only determined at 30 minutes after test substance exposure) were determined at each of the above time points.
ICE classes were determined based on a predetermined range in accordance with the criteria given in OECD TG 438.
The overall in vitro irritancy classification of the test item was determined by using all the classification information and criteria given in OECD TG 438 and the UN GHS classification referenced in the guideline. Full details are provided in the full study report.

Irritation parameter:

cornea opacity score

Run / experiment:

mean (n=3)

Value:

2.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class III

Irritation parameter:

fluorescein retention score

Run / experiment:

mean (n=3)

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class III

Irritation parameter:

percent corneal swelling

Run / experiment:

mean (n=3)

Value:

17.65

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value %; ICE Class III

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None reported.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline: Not applicable. Applicant assessment of the data indicates that the positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively.

Table 1. Ocular reactions

Test Item
Maximal mean score for corneal opacity	2.3	ICE Class III
Mean score of Fluorescein Retention	2.0	ICE Class III
Corneal swelling	17.65%	ICE Class III
Positive Control Item
Maximal mean score for corneal opacity	4.0	ICE Class IV
Mean score of Fluorescein Retention	3.0	ICE Class IV
Corneal swelling	41.94%	ICE Class IV
Negative Control Item
Maximal mean score for corneal opacity	0.5	ICE Class I
Mean score of Fluorescein Retention	0.5	ICE Class I
Corneal swelling	2.78%	ICE Class I

 

- Corneal Opacity Scores

‘Easily discernible translucent area; details of the iris are slightly obscured’ were noted in two of the test item treated eyes and ‘severe corneal opacity; no specific details of the iris are visible; size of the pupil barely discernible’ was noted in one test item treated eye. Very faint opacity was noted in the negative control treated eyes. No morphological effects were noted in the test item or negative control item treated eyes. ‘Complete corneal opacity; iris invisible’ was noted in all positive control treated eyes. Sloughing was noted in all positive control treated eyes.

- Fluorescein Retention Scores

Focal or confluent dense single cell staining was noted in the test item treated eyes. Very minor single cell staining was noted in the negative control treated eyes. Confluent large areas of the cornea retaining fluorescein were noted in all positive control treated eyes.

 

Table 2. Individual scoring - test item

End Point	Eye Number	Time (after decontamination)
		0 minutes	30 minutes	75 minutes	120 minutes	180 minutes	240 minutes
Corneal Opacity	3B	0	1	2	2	2	2
	6B	0.5	2	2	2	2	2
	8B	0	2	3	3	3	3
Mean		0.2	1.7	2.3	2.3	2.3	2.3
ICE Class		III
Fluorescein Retention	3B		2
	6B		2
	8B		2
Mean			2.0
ICE Class		III
Corneal Thickness	3B	0.72	0.84	0.86	0.80	0.84	0.84
	6B	0.75	0.80	0.78	0.90	0.88	0.84
	8B	0.74	0.82	0.84	0.88	0.88	0.88
Mean		0.74	0.82	0.83	0.86	0.87	0.85
Mean Corneal Swelling (%)			11.31	12.22	16.74	17.65	15.84
ICE Class		III
ICE Classes Combined:		3 x III
Classification:		No prediction of eye irritation can be made

 

The test was considered acceptable since the concurrent negative or vehicle/solvent items and the concurrent positive controls were identified as GHS Non Classified and GHS Category 1, respectively.

Interpretation of results:

other: no prediction can be made

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, no prediction for eye irritation can be made following assessment of the data for all endpoints.

Executive summary:

The study was performed according to OECD TG 438 in accordance with GLP to assess the eye irritancy potential of the test material in isolated chicken eyes. The method involves evaluation of the eye hazard potential of a test chemical as measured by its ability to induce toxicity in an enucleated chicken eye. Toxic effects to the cornea are measured by (i) a qualitative assessment of opacity (ii) a qualitative assessment of damage to epithelium based on application of fluorescein to the eye (fluorescein retention) (iii) a quantitative measurement of increased thickness (swelling) and (iv) a qualitative evaluation of macroscopic morphological damage to the surface. Also to identify substances not requiring UN GHS classification. 0.03 mL of the test item was applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item. A further two enucleated eyes remained untreated for control purposes. The mean corneal opacity was 2.3 (ICE Class III). The mean fluorescein retention was 2.0 (ICE Class III) and the mean corneal thickness across 30, 75, 120, 180 and 240 minutes was ICE Class III with maximal corneal swelling 17.65%. The negative control gave a prediction of GHS non-classified for eye irritation (ICE Class I) across all categories. The positive control gave a prediction of GHS Category 1 (ICE Class IV) across all categories signifying that the test system performed adequately. Under the conditions of this study, no prediction for eye irritation can be made following assessment of the data for all endpoints.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29-10-2018 to 13-11-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Justification for type of information:

Information as to the availability of the in vivo study is provided in 'attached justification'.

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: Japanese MAFF, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2017 ; signature: November 2017

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Recognised animal supplier
- Age at study initiation: 12-52 weeks old
- Weight at study initiation: 3.090 - 3.328 kg
- Housing: individually housed in suspended metal cages; with environment enrichment
- Diet: certified rabbit food ad libitum
- Water: mains water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

A volume of 0.1 mL of the test material, was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test material, and then released. The left eye remained untreated and was used for control purposes.

Observation period (in vivo):

Ocular assessment was conducted at approximately 1, 24, 48 and 72 hours after instillation of the test substance, according to numerical evaluation.

Number of animals or in vitro replicates:

2 (1 f / 1 f). Testing was conducted sequentially. Number of replcates in accordance with the relevant OECD guideline.

Details on study design:

The study was performed in a stepwise manner and was started by treatment of a single rabbit. The other animals were treated in a similar manner after considering the degree of eye irritation observed in the first and/or second animal. As applicable.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): none

SCORING SYSTEM:
The irritation was assessed according to Draize (1977) numerical scoring system. At each observation period, the highest scores given were recorded. Any other ocular effects were also noted.

TOOL USED TO ASSESS SCORE: Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: mean; n=2

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0.7

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: mean; n=2

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

1.7

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: mean; n=2

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: mean; n=2

Irritant / corrosive response data:

Scattered diffuse corneal effects were noted in both eyes up to 72 hours. iridial inflammation was noted up to 48 hours. Moderate conjunctival irritation was noted in treated eyes 1 hour after treatment. Moderate to minimal conjunctival irritation was noted at the 24 to 72 hour observations. All treated eyes appeared normal at the 7 day observation period.

Other effects:

- Lesions and clinical observations: None reported.
- Ophthalmoscopic findings: Not applicable.
- Histopathological findings: Not applicable.
- Effects of rinsing or washing: Not applicable.
- Other observations: All females gained bodyweight during the study.

Table 1. Individual scores and mean scores for 24, 48 and 72 hours

Organism number	1				2
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA
Degree of Opacity	0.0	1.0	1.0	1.0	0.0	1.0	1.0	1.0
Mean (24 – 72 h)				1.0				1.0
IRIS	0.0	1.0	1.0	0.0	0.0	1.0	1.0	0.0
Mean (24 – 72 h)				0.7				0.0
CONJUNCTIVAE
Redness	2.0	2.0	2.0	1.0	2.0	2.0	2.0	1.0
Mean (24 – 72 h)				1.7				1.7
Chemosis	2.0	1.0	1.0	1.0	2.0	1.0	1.0	1.0
Mean (24 – 72 h)				1.0				1.0

All effects reversed (score = 0) at the 7 day observation period.

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test item is not irritating to the eye.

Executive summary:

The study was performed to OECD TG 405 and EU Method B.5 under GLP to assess the irritancy potential of the test item to the eye following a single application in the New Zealand White rabbit. A volume of 0.1 ml of the test item was placed into the conjunctival sac of one eye of two animals. The other eye remained untreated and was used for control purposes. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours and/or up to 14 days following treatment, as applicable. A single application of the test item to the non-irrigated eye of two rabbits produced scattered or diffuse corneal opacity, iridial inflammation and moderate conjunctival irritation. Treated eyes appeared normal at the 7-Day observation. Under the conditions of this study, the test item is considered to be irritating to the eye.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation:

Key study : In vitro, OECD TG 439, 2018 : The study was performed to OECD TG 439 and EU Method B.46 under GLP to assess the skin irritation potential of the test item using a human three dimensional epidermal model (EPISKIN Small Model). Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 82.2% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 1.5%. The mean OD570 for the negative control treated tissues was 0.736 and the standard deviation value of the viability was 11.5%. The negative control acceptance criteria were therefore satisfied. The relative mean tissue viability for the positive control treated tissues was 9.1% relative to the negative control treated tissues and the standard deviation value of the viability was 4.9%. The positive control acceptance criteria were therefore satisfied. All acceptance criteria were considered to be met. Under the conditions of this study, the test item is not considered to be irritating to the skin.

 

Key study : In vivo, OECD TG 404, 2018 : The study was performed to OECD TG 404, EU Method B.4 and the Japanese MAFF (2000) and US EPA OPPTS 870.2500 under GLP to assess the primary skin irritancy potential of the test item in accordance with GLP in New Zealand White rabbits. Following single 4-Hour, semi-occluded applications to the intact rabbit skin. 0.5 mL of the test item was introduced under a 2.5 cm x 2.5 cm cotton gauze patch and placed in position on the clipped skin to assess the irritancy potential of the test item. The patch was secured in position with a strip of surgical adhesive tape. After 4 hours of exposure to the test item, the patches were removed and individual dose sites were scored at approximately 1, 24, 48, 72 hours and 7 days, respectively. A single 4-Hour, semi occluded application of the test item to the intact skin of three rabbits produced well-defined erythema and slight edema. Treated skin sites appeared normal at the 7-Day observation. No corrosive effects were noted. No other skin reactions noted. Mean scores for following grading at 24, 48 and 72h were 1.33, 2.0 and 2.0 in erythema and eschar and 1.0, 1.67 and 1.33 in edema scoring criteria. At day 7, mean scores were zero for erythema and edema, respectively. Under the conditions of the study, the item is not considered to be a skin irritant.

 

Eye Irritation:

Key study : In vitro, OECD TG 438, 2018 : The study was performed according to OECD TG 438 in accordance with GLP to assess the eye irritancy potential of the test material in isolated chicken eyes. The method involves evaluation of the eye hazard potential of a test chemical as measured by its ability to induce toxicity in an enucleated chicken eye. Toxic effects to the cornea are measured by (i) a qualitative assessment of opacity (ii) a qualitative assessment of damage to epithelium based on application of fluorescein to the eye (fluorescein retention) (iii) a quantitative measurement of increased thickness (swelling) and (iv) a qualitative evaluation of macroscopic morphological damage to the surface. Also to identify substances not requiring UN GHS classification. 0.03 mL of the test item was applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item. A further two enucleated eyes remained untreated for control purposes. The mean corneal opacity was 2.3 (ICE Class III). The mean fluorescein retention was 2.0 (ICE Class III) and the mean corneal thickness across 30, 75, 120, 180 and 240 minutes was ICE Class III with maximal corneal swelling 17.65%. The negative control gave a prediction of GHS non-classified for eye irritation (ICE Class I) across all categories. The positive control gave a prediction of GHS Category 1 (ICE Class IV) across all categories signifying that the test system performed adequately. Under the conditions of this study, no prediction for eye irritation can be made following assessment of the data for all endpoints.

Key study : In vivo, OECD TG 405, 2018 : The study was performed to OECD TG 405 and EU Method B.5 under GLP to assess the irritancy potential of the test item to the eye following a single application in the New Zealand White rabbit. A volume of 0.1 ml of the test item was placed into the conjunctival sac of one eye of two animals. The other eye remained untreated and was used for control purposes. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours and/or up to 14 days following treatment, as applicable. A single application of the test item to the non-irrigated eye of two rabbits produced scattered or diffuse corneal opacity, iridial inflammation and moderate conjunctival irritation. Treated eyes appeared normal at the 7-Day observation. Under the conditions of this study, the test item is considered to be irritating to the eye.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for dermal irritation

The substance meets classification criteria under Regulation (EC) No 1272/2008 for eye irritation: category 2: H319

 

For skin irritation, further in vitro skin corrosion testing does not need to be conducted based on the available information allowing a definitive conclusion on the classification of the substance. In accordance with section 1.2 of REACH Regulation (EC) No. 1907/2006 Annex XI the weight of evidence indicates that the substance is not skin corrosive and therefore in vitro testing may be omitted. An available in vitro (OECD TG 439, 2018) skin irritation assay indicates that the substance is not irritating to the skin (based on > 60% MTT viability). An available in vivo irritation study (rabbit: OECD TG 404, 2018) indicates the erythema and edema scores were not sufficient for classification (< 2.3 mean scoring 24 to 72 hours in 2/3 organisms) and are suggestive of Globally Harmonised System (revision 4): Skin Irritation category 3 - mild irritant.

 

For eye irritation, the available in vitro assay (ICE, OECD TG 438, 2018) could not make a prediction of irritation. Based on an available in vivo study (OECD TG 405, 2018) where the mean scoring and evaluation of the results where two of three organisms demonstrated that the EU criteria had been met: the substance has the potential to cause transient irritating effects to the eye which are sufficient for classification.

 

References:

1. ECHA Guidance on Application on the CLP Criteria, (v5.0, July 2017)