Registration Dossier

Administrative data

Description of key information

The skin irritation potential of the test item was evaluated using EPISKIN (TM) reconstructed human epidermis model and no evidence of corrosivity or classifiable skin irritation was observed. The in vitro eye irritation study on the enucleated rabbit eye indicated that the test material was unlikely to be irritant or corrosive to the eye. The in vivo study showed the material to be mildly irritant to rabbit eyes but below the threshold for classification.

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:
28 August 2101 to 03 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 439 (In Vitro Skin Irritation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Not reported
Source strain:
not specified
Details on animal used as source of test system:
- Source: EPISKIN (TM) model kit
- Supplier: Skin Ethic Laboratories, Nice, France
Justification for test system used:
The EPISKIN model is a three-dimernsional 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.
Vehicle:
unchanged (no vehicle)
Details on test system:
The study was carried on a 50% w.w. concentration of the test item in pharmaceutical white oil.
To make sure that the test item did not directly reduce the MTT to blue formazan, 10 µL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37°C and 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control. As the MTT solution containing the test item did not turned blue the test item was shown not to directly reduce MTT.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
- Treatment group: Test carried out in triplicate. 10 µL of the test item was applied topically, ensuring an even covering, to the epidermis surface.
Duration of treatment / exposure:
- Treatment period: 15 minutes
Duration of post-treatment incubation (if applicable):
- Post-exposure incubation: At the end of the exposure period, tissues were rinsed with DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a soft stream of DPBS to gently remove any residual test item. Residual test item remained on the tissue surface after rinsing. The rinsed tissues were transferred to a second column of three wells, each containing 2 mL of maintenance medium, and incubated for 42 hours, at 37°C and 5% CO2 in air.
Number of replicates:
Triplicate samples of epidermis tissue were uniformly covered with approximately 10 µL of test item. At the end of the 15 minute exposure period, each tissue was removed and rinsed with DPBS with Ca++ and Mg++. Residual test item remained on the tissue surface after rinsing. The tissues were then incubated for 42 hours at 37°C and 5% CO2 in air, in wells containing 2 mL maintenance media.
Details on study design:
The measurement of tissue viability (cytotoxicity) was measured by means of the colourimetric MTT reduction assay. Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt (by the mitochondrial succinate dehdroganase in viable cells) in the test item treated tissue relative to the negative control using the following procedure. After incubation, the triplicate tissue samples were transferred into three wells each containing 2ml of a 0.3 mg/L MTT solution, care being taken to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37°C, 5% CO2 in air and then the epidermis was carefully separated from the collagen matrix using forceps and both parts placed into 1.5 mL microtubes containing 500 µL of acidified isopropanol. Each tube was plugged and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°C for 3 days to allow the extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was thoroughly mixed on a vortex mixer to produce a homogeneous coloured solution. The optical density of the extracted solution was measured at 540 nm against an acidified isopropanol blank.
The relative mean viability were calculated using the following equation: Relative mean viability (%) = (mean OD540 of test item/mean OD540 of negative control) x 100. If the mean tissue viability is ≤50%, the substance is considered to be irritant.
Irritation / corrosion parameter:
other: other: Percent Relative Viability
Value:
93
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Basis: mean. Time point: After a 15 minute exposure. Max. score: 94.9. Reversibility: No data.
Other effects / acceptance of results:
- Remarks: Negative control item (set at 100). Standard deviation relative to mean viability ± 3.0. (migrated information)
- Viability: The relative mean viability of the treated tissues was 93.0 % after a 15-minute exposure period, with standard deviation of 3.0 %.
- Optical density: The mean optical density of the treated tissues at 540 nm was 0.735
- Direct MTT reduction: The MTT solution containing the test item did not turn blue, indicating that test item did not directly reduce MTT.
- Positive control: The relative mean tissue viability was 11.8 % relative to the negative control and the standard deviation was 3.8%
- Negative control: The mean optical density was 0.790 and the standard deviation was 0.046.
- Acceptance criteria: Satisfied according to the protocol criteria for both the positive control (relative mean viability ≤ 40% and standard deviation ≤ 18%) and negative control (OD540 ≥ 0.6 and standard deviation of individual tissue viability ≤18%). The standard deviation of the triplicate treated tissues was 3.0 % and hence the test item acceptance criterion (≤ 18%) was also satisfied.
- Conclusion: Test item is considered to be non-irritant using the EPISKIN (TM) human epidermis model (viability >50%)

Table 1. Mean OD540Values and Percentage Viability for Negative Control Item, Positive Control Item and Test Item

Item

OD540of tissue

Mean OD540of triplicate tissues

±SD of OD540

Relative Individual tissue viability (%)

Relative mean viability (%)

±SD of Relative mean viability (%)

Negative Control Item1

0.843

0.790

0.046

106.7

100*

5.5

0.758

97.2

0.760

96.2

Positive Control Item1

0.119

0.093

0.030

15.1

11.8

3.8

0.100

12.7

0.060

7.6

Test Item

0.708

0.735

0.023

89.6

93.0

3.0

0.747

94.6

0.750

94.9

SD = Standard deviation

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

1Control group shared with test laboratory project number 41202604

Interpretation of results:
other: Criteria for classification as a skin irritant not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The substance is considered to be non-irritant using the EPISKIN (TM) human epidermis model.
Executive summary:

The substance is considered to be non-irritant using the EPISKIN (TM) human epidermis model. The skin irritation potential of the test item was evaluated using EPISKIN (TM) reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure period of 42 hours using a colourimetric MTT reduction assay following OECD guideline 439 in an experimental proprietary study (Harlan 2013). The quality criteria required for the acceptance of results in the test were satisfied and the study is considered reliable and relevant for use. The study was carried on a 50% w.w. concentration of the test item in pharmaceutical white oil.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 November 2012 to 22 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
The study was carried on a 50% w.w. concentration of the test item in pharmaceutical white oil.
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Two New Zealand White rabbits were supplied by Harlan Laboratories UK Limited, Hillcrest, Belton, Loughborough, UK. At the start of the study the animals weighed 2.59 or 2.93 kg and were twelve to twenty weeks old. After an acclimatisation period of at least five days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2030C Teklad Global Rabbit diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 17 to 23°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
A volume of 0.1 mL of the test material was placed into the conjunctival sac of the right eye.
Duration of treatment / exposure:
Up to 72 hours
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
2
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: Not applicable
- Procedure: Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used. Initially, a single rabbit was treated. A volume of 0.1 mL of the test item 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. Immediately after administration of the test material, an assessment of the initial pain reaction was made according to the six point scale shown in Appendix 1. After consideration of the ocular responses produced in the first treated animal, a second animal was treated. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation given in Appendix 2, (from Draize J H (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC p.48 to 49).
- Assessment of effects: Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope. Any clinical signs of toxicity, if present, were also recorded. Individual bodyweights were recorded on Day 0 (the day of dosing) and at the end of the observation period.
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal: 72664 Male
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 48 hours
Remarks on result:
other: Highest score observed at 1 and 24 hour timepoints
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal: 72702 Male
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 48 hours
Remarks on result:
other: Highest score observed at 1 hour and 24 hour timepoints
Irritation parameter:
chemosis score
Basis:
animal: 72664 Male
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 48 hours
Remarks on result:
other: Highest score observed at 1 hour timepoint
Irritation parameter:
chemosis score
Basis:
animal: 72702 Male
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
fully reversible within: 24 hours
Remarks on result:
other: Highest score observed at 1 hour timepoint
Irritation parameter:
cornea opacity score
Basis:
animal: 72664 Male
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: No effect noted
Irritation parameter:
cornea opacity score
Basis:
animal: 72702 Male
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: No effect noted
Irritation parameter:
iris score
Basis:
animal: 72664 Male
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: No effect noted
Remarks on result:
other: Mean
Irritation parameter:
iris score
Basis:
animal: 72702 Male
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: No effect noted
Irritation parameter:
other: Conjunctivae Discharge
Basis:
animal: 72664 Male
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: No effects noted
Irritation parameter:
other: Conjunctivae Discharge
Basis:
animal: 72702 Male
Time point:
24/48/72 h
Score:
0
Max. score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
other: Highest score observed at 1 hour timepoint
Irritant / corrosive response data:
Individual and group mean scores for ocular irritation are given in Table 1 and Table 2 in any other information on results section. Individual mean scores for cornea, iris and conjunctivae are given in Table 3.
No corneal or iridial effects were noted during the study.
Moderate conjunctival irritation was noted in one treated eye with minimal conjunctival irritation noted in the other treated eye one hour after treatment. Minimal conjunctival irritation was noted in both treated eyes at the 24 Hour observation.
Both treated eyes appeared normal at the 48 Hour observation.
Other effects:
Both animals showed expected gain in bodyweight during the study (Table 4).

Table 1              Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex

72664 Male

72702 Male

IPR = 0

IPR = 0

Time After Treatment

1
Hour

24
Hours

48
Hours

72
Hours

1
Hour

24
Hours

48
Hours

72
Hours

CORNEA

 

 

 

 

 

 

 

 

E = Degree of Opacity

0

0

0

0

0

0

0

0

F = Area of Cornea Involved

0

0

0

0

0

0

0

0

Score (E x F) x 5

0

0

0

0

0

0

0

0

IRIS

 

 

 

 

 

 

 

 

D

0

0

0

0

0

0

0

0

Score (D x 5)

0

0

0

0

0

0

0

0

CONJUNCTIVAE

 

 

 

 

 

 

 

 

A = Redness

1

1

0

0

1

1

0

0

B = Chemosis

2

1

0

0

2

0

0

0

C = Discharge

0

0

0

0

1

0

0

0

Score (A + B + C) x 2

6

4

0

0

8

2

0

0

Total Score

6

4

0

0

8

2

0

0

IPR=  Initial pain reaction

Interpretation According to the Globally Harmonized System of Classification and Labelling of Chemical

Irreversible eye effects categories

An eye irritant Category 1 (irreversible effects on the eye) is a test material that produces:

-

at least in one animal effects on the cornea, iris or conjunctiva that are not expected to reverse or have not fully reversed within an observation period of normally 21 days; and/or

-

at least in 2 of 3 tested animals a positive response of:

corneal opacity³ 3 and/or

iritis³ 1.5r

calculated as the mean scores following grading at 24, 48 and 72 hours after instillation of the test material

Reversible eye effects categories

An eye irritant Category 2A (irritating to eyes) is a test material that produces:

-

at least in 2 of 3 tested animals a positive response of:

corneal opacity³ 1 and/or

iritis³ 1 and/or

conjunctival redness³ 2 and/or

conjunctival oedema (chemosis)³ 2

-

Calculated as the mean scores following grading at 24, 48 and 72-Hour observations after instillation of the test material, and

at

-

which fully reverses within an observation period of normally 21 days

Within this category an eye irritant is considered mildly irritating to eyes (Category 2B) when the effects listed above are fully reversible within 7 days of observation.

Interpretation According to Regulation (EC) No 1272/2008, Relating to the Classification, Labelling and Packaging of Dangerous Substances

        

Category for irreversible eye effects

Category

Criteria

Irreversible effects on the eye

(Category 1)

If, when applied to the eye of an animal, a substance produces:

-  at least in one animal effects on the cornea, iris or conjunctiva that   are not expected to reverse or have not fully reversed within an observation period of normally 21 days;

and/or

-     at least in 2 of 3 animals, a positive response of:

-     cornea opacity³3 and/or

-     iritis>1.5

calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material.

Category for reversible eye effects

Category

Criteria

Irritating to eyes

(Category 2)

If, when applied to the eye of an animal, a substance produces:

-     at least in 2 of 3 tested animals, a positive response of:

-   cornea opacity³1 and/or

-   iritis>1, and/or

-   conjunctival redness³2 and/or

-   conjunctival oedema (chemosis)³2

-   calculated as the mean scores following grading at 24, 48 and         72 hours after installation of the test material, and which fully reverses within an observation period of 21 days.

For those substances where there is pronounced variability among animal responses, this information shall be taken into account in determining the classification.

Table 2              Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbit Number

and Sex

Individual Total Scores At:

1 Hour

24 Hours

48 Hours

72 Hours

72664 Male

6

4

0

0

72702 Male

8

2

0

0

Group Total

14

6

0

0

Group Mean Score

7.0

3.0

0.0

0.0

Table 3            Individual and Mean Scores for Cornea, Iris and Conjunctivae

Rabbit Number and Sex

Time After Treatment

Corneal Opacity

Iridial Inflammation

Conjunctival Redness

Conjunctival Chemosis

72664 Male

24 Hours

0

0

1

1

48 Hours

0

0

0

0

72 Hours

0

0

0

0

Total

0

0

1

1

Mean

0.0

0.0

0.3

0.0

72702 Male

24 Hours

0

0

1

1

48 Hours

0

0

0

0

72 Hours

0

0

0

0

Total

0

0

1

1

Mean

0.0

0.0

0.3

0.0

 

Table 4              Individual Bodyweights and Bodyweight Changes

Rabbit Number
and Sex

Individual Bodyweight (kg)

Bodyweight Change (kg)

Day 0

Day 3

72664 Male

2.59

2.67

0.08

72702 Male

2.93

2.98

0.06

Interpretation of results:
other: Criteria for classification as an eye irritant not met
Remarks:
Criteria used for interpretation of results: Kay and Calandra classification
Conclusions:
The test material produced a maximum group mean score of 7.0 and was classified as a minimal irritant (Class 3 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system. The substance does not meet the criteria for classification according to the Globally Harmonised System of Classification and Labelling of Chemicals or according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Dangerous Substances.
Executive summary:

Introduction.

The study was performed to assess the irritancy potential of the test material to the eye of the New Zealand White rabbit. The assessment was carried out in a GLP-compliant study following OECD Guidelines for the Testing of Chemicals No. 405 “Acute Eye Irritation/Corrosion” (adopted 24 April 2002) and Method B5 Acute Toxicity (Eye Irritation) of Commission Regulation (EC) No. 440/2008 in a proprietary, experimental study (Harlan 2013). The study is considered reliable and relevant for use for this endpoint.

Result.

A single application of the test material to the non-irrigated eye of two rabbits produced moderate conjunctival irritation in one treated eye and minimal conjunctival irritation in the other treated eye one hour after treatment with minimal conjunctival irritation noted in both treated eyes at the 24-hour observation. Both treated eyes appeared normal at the 48-hour observation.

Conclusion.

The test material produced a maximum group mean score of 7.0 and was classified as a minimal irritant (Class 3 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system. The substance does not meet the criteria for classification according to the Globally Harmonised System of Classification and Labelling of Chemicals or according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Dangerous Substances.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Principles of method if other than guideline:
The rabbit enucleated eye test is used as a first stage in the assessment of ocular irritancy potential. The assay has undergone inter-laboratory validation and has been shown to reliably detect test items that are negligible, or moderate to severe ocular irritants.
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
not specified
Details on test animals or tissues and environmental conditions:
- State of eyes: Enucleated
- Selection of eyes: Prior to enucleation, the eyes of the provisionally selected rabbits were examined for evidence of ocular irritation or defect, following application of Fluorescein Sodium drops BP (1% w/v). Corneal thickness values were recorded. Only animals whose eyes showed no evidence of ocular irritation or defect were used for testing purposes (see below).
- Enucleation of eyes: Rabbits were sacrificed and two to three drops of saline solution at approximately 32°C were immediately applied to the cornea. The eye was carefully removed, positioned in a perspex clamp and placed within the superfusion chamber, with the surface irrigated by a saline drip. The eyes were then allowed to equilibrate for approximately thirty minutes, before being re examined and the corneal thickness measured. Any eyes in which the corneal swelling was greater than 10% relative to the pre-enucleation measurement, or in which the cornea was stained with fluorescein, were rejected.
- Environmental conditions: The superfusion apparatus was set to a stable temperature of 32 ±1.5°C within the chamber and a peristaltic pump supplied saline solution at approximately 32°C at a flow rate of 0.15 to 0.4 mL/minute.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: The test item was used undiluted as supplied. A volume of 0.1 mL of the test item was applied evenly as possible to the surface of the cornea.
- Concentration: Substance tested as 50% ww concentration in pharmaceutical white oil (CAS No. 8042-47-5)
Duration of treatment / exposure:
- Exposure: Ten seconds, then the test item was washed off the cornea using a minimum of 20 mL of saline solution at approximately 32°C
Observation period (in vivo):
- Assessment of corneal cloudiness: Assessment was made pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment
- Measurements for corneal thickness: Pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment
- The condition of the corneal epithelium: Approximately 60, 120, 180 and 240 minutes following treatment
- The uptake of fluorescein by the corneal epithelium: Pre-enucleation, post equilibration and approximately 240 minutes following treatment
Number of animals or in vitro replicates:
- Number of eyes: Three eyes were treated with test item, two additional eyes remained untreated for control purposes.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: Yes, the test item was washed off the cornea using a minimum of 20 mL of saline solution (approximately 32ºC)
- Time after start of exposure: Ten seconds

SCORING SYSTEM: Assessment of corneal cloudiness was made according to the numerical evaluation adopted from Advances in Modern Toxicology: Dermatoxicology, 4th Ed, (F Marzulli and H Maibach, eds) Hemisphere Publishing Corporation, Washington DC, 1991, pp 749-815.

TOOL USED TO ASSESS SCORE: A slit-lamp biomicroscope was used to examine the eye and the thickness of the cornea was measured using an ultrasonic pachymeter. For each enucleated eye a measurement was made at the optical centre, and at a further four locations at the apex of the cornea.
- Statistics: A mean value for corneal thickness was calculated from the values for the optical centre and the apex.
Irritation parameter:
cornea opacity score
Run / experiment:
Mean
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Irritation parameter:
fluorescein retention score
Run / experiment:
Mean
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Other effects / acceptance of results:
- Corneal opacity: No corneal effects were noted in the test eyes or control eyes during the study period (Table 1)
- Corneal thickness: No corneal opacities or corneal swelling observed. Corneal swelling of the test eyes during the study period was comparable to that observed in the control eyes over the same period (Table 2 and Table 3)
- Corneal condition: The condition of the corneal epithelium of the test eyes and control eyes appeared normal during the study period (Table 4)
- Fluorescein uptake: No fluorescein uptake was noted in the test eyes or control eyes 240 minutes after treatment (Table 5)
Other effects:
- Conclusion: Following assessment of the data for all endpoints, the test item was considered unlikely to have the potential to cause severe ocular irritancy in vivo

Table1              Individual Scores for Corneal Opacity

 

Test Eyes

Control Eyes

Chamber Number

1

3

5

2

4

Time After Treatment (minutes)

60

120

180

240

60

120

180

240

60

120

180

240

60

120

180

240

60

120

180

240

Degree of Corneal Opacity

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Area of Corneal Opacity

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Maximum Corneal Opacity (corneal cloudiness x area)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Table2              Individual Measurements for Corneal Thickness (µm)

Test Eyes

Time After Treatment (minutes)

60

120

180

240

Corneal Position

1·

2

3

4

5

mean

1·

2

3

4

5

mean

1·

2

3

4

5

mean

1·

2

3

4

5

mean

Chamber Number

1

437

 456

433

416

418

432.0

432

431

414

399

410

417.2

420

412

396

395

390

402.6

405

407

402

391

384

397.8

3

443

451

452

426

443

443.0

444

443

458

437

446

445.6

436

437

438

419

430

432.0

417

439

423

399

432

422.0

5

428

408

396

403

396

406.2

418

397

388

381

387

394.2

411

392

395

388

380

391.2

415

386

387

385

383

391.2

 

Control Eyes

Time After Treatment (minutes)

60

120

180

240

Corneal Position

1·

2

3

4

5

mean

1·

2

3

4

5

mean

1·

2

3

4

5

mean

1·

2

3

4

5

mean

Chamber Number

2

444

419

433

439

424

431.8

432

395

417

431

406

416.2

411

392

403

419

403

405.6

414

391

393

419

391

401.6

4

427

394

429

423

427

420.0

427

400

422

428

425

420.4

415

395

429

428

427

418.8

410

401

420

427

419

415.4

·=       Optical centre

Table3              Determination of Corneal Swelling (%)

Test Eyes

Chamber Number

Observation Period (minutes)

Mean Corneal Thickness (µm)

Corneal Swelling (%)a

Chamber Number

Observation Period (minutes)

Mean Corneal Thickness (µm)

Corneal Swelling (%)a

Chamber Number

Observation Period (minutes)

Mean Corneal Thickness (µm)

Corneal Swelling (%)a

1

Post equilibration

409.0

N/A

3

Post equilibration

399.0

N/A

5

Post equilibration

385.0

N/A

60 Post treatment

432.0

5.6

60 Post treatment

443.0

11.0

60 Post treatment

406.2

5.5

120 Post treatment

417.2

2.0

120 Post treatment

445.6

11.7

120 Post treatment

394.2

2.4

180 Post treatment

402.6

0.0

180 Post treatment

432.0

8.3

180 Post treatment

391.2

1.6

240 Post treatment

397.8

0.0

240 Post treatment

422.0

5.8

240 Post treatment

391.2

1.6

 

Control Eyes

 

Chamber Number

Observation Period (minutes)

Mean Corneal Thickness (µm)

Corneal Swelling (%)a

Chamber Number

Observation Period (minutes)

Mean Corneal Thickness (µm)

Corneal Swelling (%)a

Test Eyes

Mean corneal swelling 1 hour following treatment 7.4%

Mean corneal swelling 2 hours following treatment 5.4%

Mean corneal swelling 4 hours following treatment 2.5%

2

Post equilibration

399.4

N/A

4

Post equilibration

396.0

N/A

60 Post treatment

431.8

8.1

60 Post treatment

420.0

6.1

Control Eyes

120 Post treatment

416.2

4.2

120 Post treatment

420.4

5.2

Mean corneal swelling 1 hour following treatment 7.1%

Mean corneal swelling 2 hours following treatment 5.2%

Mean corneal swelling 4 hours following treatment 2.7%

180 Post treatment

405.6

1.6

180 Post treatment

418.8

5.8

240 Post treatment

401.6

0.6

240 Post treatment

415.4

4.9



a=     % Corneal swelling = (mean corneal thickness post-treatment) – (mean corneal thickness post equilibratiom)x 100

                                               Mean corneal thickness post equilibration

N/A=  Not applicable

Table 4              Corneal Epithelium Condition

Test Eyes

Chamber Number

Time After Treatment (minutes)

60

120

180

240

1

Normal

 Normal

 Normal

  Normal

3

 Normal

 Normal

 Normal

 Normal

5

 Normal

 Normal

 Normal

 Normal

 

Control Eyes

Chamber Number

Time After Treatment (minutes)

60

120

180

240

2

 Normal

 Normal

 Normal

 Normal

4

 Normal

 Normal

 Normal

 Normal

Table5              Individual Scores for Fluorescein Uptake (240 Minutes Post Dosing)

 

Test Eyes

Control Eyes

Chamber Number

1

3

5

2

4

Intensity of Fluorescein Uptake

0

0

0

0

0

Area of Fluorescein Uptake

0

0

0

0

0

Maximum Fluorescein Uptake
(intensity x area)

0

0

0

0

0

 

Interpretation of results:
other: Unlikely to have the potential to cause severe ocular irritancy in vivo
Conclusions:
No corneal effects or fluorescein uptake were noted, corneal swelling of the test eyes was comparable to the control eyes and the condition of the corneal epithelium appeared normal. Following assessment of the data for all endpoints, the substance was considered unlikely to have the potential to cause severe ocular irritancy in vivo.
Executive summary:

The substance was considered unlikely to have the potential to cause severe ocular irritancy in vivo. The occular irritation potential of the test item was assessed in a GLP-compliant, non-guideline study on enucleated rabbit eyes (Harlan 2013). The test item was applied to the cornea of three enucleated rabbit eyes maintained at 32°C in a superfusion chamber and maximal ocular irritation observations were recorded at 60, 120 and 240 minutes, for corneal opacity, fluorescein uptake and condition of the corneal epithelium. The study is considered reliable and relevant for use for this endpoint.

The study was conducted on a 50% w.w. concentration of the test item in pharmaceutical white oil.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The skin irritation potential of the test item was evaluated using the in vitro EPISKIN (TM) reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure period of 42 hours using a colourimetric MTT reduction assay following OECD guideline 439 in an experimental proprietary study. The quality criteria required for the acceptance of results in the test were satisfied and the study is considered reliable and suitable for use. No evidence of corrosivity or classifiable skin irritation was observed.

The occular irritation potential of the test item was first assessed in a GLP-compliant, non-guideline in vitro study on enucleated rabbit eyes. The test item was applied to the cornea of three enucleated rabbit eyes maintained at 32°C in a superfusion chamber and maximal ocular irritation observations were recorded at 60, 120 and 240 minutes, for corneal opacity, fluorescein uptake and condition of the corneal epithelium. The study is considered reliable and suitable for use for this endpoint. No evidence of corrosion was observed.

An in vivo study was performed to assess the irritancy potential of the test material to the eye of the New Zealand White rabbit. The assessment was carried out in a GLP-compliant study following OECD Guidelines for the Testing of Chemicals No. 405 “Acute Eye Irritation/Corrosion” (adopted 24 April 2002) and Method B5 Acute Toxicity (Eye Irritation) of Commission Regulation (EC) No. 440/2008 in a proprietary, experimental study. The study is considered reliable and suitable for use for this endpoint. No evidence of classifiable eye irritation was observed.

The studies were carried out on a 50% w.w. concentration of the test item in pharmaceutical white oil.


Justification for selection of skin irritation / corrosion endpoint:
Only one in vitro study available using human skin cells.

Justification for selection of eye irritation endpoint:
The in vivo GLP study in rabbits has been taken as representative of eye irritation assessment.

Justification for classification or non-classification

The substance is considered to be non-irritant to skin using the EPISKIN (TM) human epidermis model.

The substance is considered not to meet the criteria for classification as irritating to eyes according to the Globally Harmonised System of Classification and Labelling of Chemicals or according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Dangerous Substances.