Fatty acids C16-18 (even numbered), mono and di esters with Sucrose

Endpoint summary

Description of key information

Skin irritation/corrosion (OECD 404): not irritating

Eye irritation: (WoE: OECD 405, OECD 437 and OECD 438 ): irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Jan - 28 Jan 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted 24 April 2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Directive (EC) No 2004/73/EC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Agence française de sécurité sanitaire des produits de santé (afssaps), France

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elevage de Gerome, Linxe, France
- Weight at study initiation: 2.39 - 2.84 kg
- Housing: individually
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 21
- Humidity (%): 30 - 32

Type of coverage:

semiocclusive

Preparation of test site:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated area on left flank served as control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 g

Duration of treatment / exposure:

4 h

Observation period:

72 h

Number of animals:

3
Initially, a single animal was treated. After consideration of the cutaneous responses produced in the first treated animal, two additional animals were treated.

Details on study design:

TEST SITE
- Area of exposure: undamaged skin area of the right flank of each animal
- Type of wrap if used: The patch was secured in position with a strip of surgical adhesive tape.

REMOVAL OF TEST SUBSTANCE
- Washing: not specified

OBSERVATION TIME POINTS
1, 24, 48 and 74 h

SCORING SYSTEM:
- Method of calculation: Draize scoring system

Irritation parameter:

erythema score

Basis:

mean

Remarks:

of 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

edema score

Basis:

mean

Remarks:

of 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritant / corrosive response data:

No cutaneous reactions (erythema and oedema) were observed at any observation time point (1, 24, 48 and 72 hours).

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the present skin irritation test in rabbits the test substance shows no skin irritating potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

21 May - 11 Jun 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted Feb 24, 1987

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Remarks:

Chbb:NZW (SPF)

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Chemical pharmaceutical factory Dr. K. Thomae, Biberach, Germany
- Weight at study initiation: 2.7 - 3.2 kg
- Housing: individually
- Diet: ssniff® K-H (V2333), ad libitum and hay (approx. 15 g daily)
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): fully air-conditioned
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 100 mg

Duration of treatment / exposure:

24 h

Observation period (in vivo):

7 days
Reading time points: 1, 24, 48 and 72 h

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: Treated eyes were washed out thoroughly with isotonic saline at approx. 37 °C.
- Time after start of exposure: 24 h after exposure and at all other designated examination times at which the treated eyes still showed discharge or at which a corneal examination with fluorescein sodium solution took place

SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

2

Reversibility:

fully reversible within: 48 h

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

out of 2 animals

Time point:

24/48/72 h

Score:

2.7

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2.3

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

out of all 3 animals

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritant / corrosive response data:

One hour up to 3 days after application the conjunctivae of the animals showed an evident hyperaemia of the blood vessels to a diffuse beefy red and a swelling with lids about half closed. The iris was reddened in 2/3 animals. Diffuse to translucent areas of the cornea were observed in the animals one day up to 3 days after application. In addition a clear colourless or white-yellowish viscous discharge and blanched nictating membrane occurred in the animals. 7 days after application no more signs were seen, indicating full reversibility.

Table 1. Results of the eye irritation study

Alteration	Animal No.	Time (h)
		24	48	72	mean 24/48/72 h	reversible within
Cornea	1	2	2	2	2.0	7 days
	2	1	0	0	0.3	48 h
	3	2	1	1	1.3	7 days
Iris	1	1	1	1	1.0	7 days
	2	1	0	0	0.3	48 h
	3	0	0	0	0.0	n.a.
Conjunctivae Redness	1	3	3	2	2.7	7 days
	2	3	3	1	2.3	7 days
	3	3	3	2	2.7	7 days
Conjunctivae Chemosis	1	3	0	0	1.0	72 h
	2	2	1	0	1.0	72 h
	3	2	1	0	1.0	72 h

Interpretation of results:

other: Eye Irrit. 2 according to Regulation (EC) 1272/2008

Conclusions:

Under the conditions of this eye irritation study in rabbits the test substance was irritating to the eye.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

17 Feb - 07 Mar 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted Feb 24, 1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 92/69/EEC, Dez 1992

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Remarks:

(SPF)

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Broekman Institute, Someren, Netherlands
- Age at study initiation: 8 or 9 weeks
- Housing: individually in cages with perforated floors
- Diet: Standard laboratory rabbit diet (LKK-20, pellet diameter 4 mm, Hope Farms, Woerden, Netherlands), approx. 100 g per day; hay (BMI, Helmond, Netherlands) once a week
- Water: tap water diluted with decalcified water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 50
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 87.8 ± 4.0 mg (approx. 0.1 mL)

Duration of treatment / exposure:

24 h

Observation period (in vivo):

14 days
Reading time points: 1, 24, 48 and 72 h, 7 and 14 days

Number of animals or in vitro replicates:

3 males

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: both eyes of the animal were rinsed with approx 50 mL tepid tap-water per eye, using a velocity of flow which did not affect the eyes, to remove residual test substance.
- Time after start of exposure: 24 h, after fluorescein examination

SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

of 2 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

2

Reversibility:

fully reversible within: 48 h

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.7

Max. score:

2

Reversibility:

fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

out of 2 animals

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 7 or 14 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.7

Max. score:

3

Reversibility:

fully reversible within: 14 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

out of 2 animals

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 72 h or 7 days

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.7

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

Two animals showed slight dulling of the normal lustre of the cornea (opacity grade 0) 1-24 hours after instillation. The third animal showed corneal injury consisting of opacity (maximum grade 1) and epithelial damage (maximum 50% of the corneal area). The corneal injury in this animal had resolved within 7 days. Iridic irritation grade 1 was observed in two animals and had resolved within 48-72 hours. The irritation of the conjunctivae consisted of redness, chemosis and discharge and had completely resolved within 7 days in one animal and within 14 days in the other two animals.
There was no evidence of severe eye damages.

Other effects:

- Lesions and clinical observations: Reduced elasticity of the eyelids was observed in two animals 48 and 72 hours after instillation.
- Other observations: No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Table 1. Results of the acute eye irritation test

Alteration	Animal No.	Time (h)
		24	48	72	mean 24/48/72 h	reversible within
Cornea	1	0	0	0	0	n.a.
	2	0	0	0	0	n.a.
	3	1	1	1	1.0	7 d
Iris	1	1	0	0	0.3	48 h
	2	0	0	0	0	n.a.
	3	1	1	0	0.7	72 h
Conjunctivae Redness	1	2	3	1	2.0	14 d
	2	2	2	2	2.0	7 d
	3	3	3	2	2.7	14 d
Conjunctivae Chemosis	1	3	1	0	1.3	72 h
	2	2	1	1	1.3	7 d
	3	3	1	1	1.7	7 d

n.a.: not applicable

Interpretation of results:

other: Eye Irrit. 2 according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of this eye irritation study in rabbits the test substance was irritating to the eye.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24 Jan - 23 Feb 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Hokkaido Livestock Corporation, Douo Plant, Hayakita Factory
- Characteristics of donor animals: The eyeballs of healthy cattle aged 12 months or older and less than 60 months were used.
- Storage, temperature and transport conditions of ocular tissue: The eyeballs collected were immersed in Hanks' balanced salt solution, and stored and transported at less than 20 °C until use in the test operation (temperature range during transportation: 9 °C to 11 °C).
- Time interval prior to initiating testing: The corneas were isolated on the same day after delivery of the eyes and directly used in the BCOP test.
- Indication of any existing defects or lesions in ocular tissue samples: The eyeballs were macroscopically examined for defects including corneal opacity, scratches, and neovascularization, and only corneas free of these abnormalities were used in the study.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: 20% (w/v) in physiological saline

VEHICLE
- Amount applied: 0.75 mL
- Lot/batch no.: K6F83

POSITIVE CONTROL
- Amount applied: 0.75 mL
- Concentration: 20% (w/v) in physiological saline
- Lot/batch no: LAP7185

Duration of treatment / exposure:

4 h

Number of animals or in vitro replicates:

triplicates for each treatment and control group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
The corneas were dissected with a 2 to 3 mm rim of the sclera remaining, and were immersed in Hanks' balanced salt solution. Isolated corneae were mounted in corresponding corneal holders consisting of anterior and posterior chambers, which interfaced with the epithelial and endothelial sides of the cornea, respectively. Both chambers were filled with phenol red free MEM, and then the corneas were immersed in the medium and equilibrated in an incubator set at 32°C ± 1 °C for 1 hr.

QUALITY CHECK OF THE ISOLATED CORNEAS:
Following the equilibration, the medium was collected and removed from both chambers of the corneal holders, and the chambers were filled with fresh phenol red free MEM. The baseline opacity value of each cornea was measured using the opacitometer (BASF-OP3.0, BASF).
The cornea was then macroscopically examined again for defects including opacity, scratches, and neovascularization. Corneas with abnormalities or opacity greater than 7 opacity units were not used in the study.

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE:
The test substance was rinsed off from the application side with phenol red-containing MEM for 3 times.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of UV/VIS spectrophotometry (OD490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS), IVIS = mean opacity value + (15x OD490 value)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as serious eye damage and labelled Category 1 according to CLP/EPS/GHS.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55 no prediction can be made.

Irritation parameter:

in vitro irritation score

Remarks:

mean of 3 corneas

Run / experiment:

4 h

Value:

13.8

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Remarks:

negative control is a vehicle control

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No abnormalities (scratches, neovascularization, etc.) were macroscopically observed in any cornea in the process of measurement.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: The opacity (0.0) and permeability (0.008) values of the vehicle control were less than the upper limits (opacity, 2.4; permeability, 0.027) of the historical control opacity and permeability values.
- Acceptance criteria met for positive control: The IVIS value of the positive control (100.4) was within 2 standard deviations (57.4 to 137.0) of the latest historical control mean.

Interpretation of results:

other: no prediction can be made

Conclusions:

The irritation potential of the test substance was assessed in the BCOP assay. Application of a 20% solution of the test substance in saline to bovine corneas resulted in a calculated mean IVIS of 13.8. According to OECD Guideline 437, since this value is in the range of >3 to <=55, no prediction on the irritation potential of the test substance can be made.

Reference 4

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

(2013)

Deviations:

no

GLP compliance:

yes

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Hokkaido Livestock Corporation, Douo Plant, Hayakita Factory
- Characteristics of donor animals: The eyeballs of healthy cattle aged 12 months or older and less than 60 months were used.
- Storage, temperature and transport conditions of ocular tissue: The eyeballs collected were immersed in Hanks' balanced salt solution, and stored and transported at less than 20 °C until use in the test operation (temperature range during transportation: 7 °C to 18 °C).
- Time interval prior to initiating testing: The corneas were isolated on the same day after delivery of the eyes and directly used in the BCOP test.
- Indication of any existing defects or lesions in ocular tissue samples: The eyeballs were macroscopically observed for defects including corneal opacity, scratches, and neovascularization, and only corneas free of these abnormalities were used in the study.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: 20% (w/v) in physiological saline

VEHICLE
- Amount applied: 0.75 mL
- Lot/batch no.: K5A82

POSITIVE CONTROL
- Amount applied: 0.75 mL
- Concentration: 20% (w/v) in physiological saline
- Lot/batch no.: LAM7343

Duration of treatment / exposure:

4 h

Number of animals or in vitro replicates:

triplicates for each treatment and control group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
The corneas were dissected with a 2 to 3 mm rim of the sclera remaining, and were immersed in Hanks' balanced salt solution. Isolated corneas were mounted in corresponding corneal holders consisting of anterior and posterior chambers, which interfaced with the epithelial and endothelial sides of the cornea, respectively. Both chambers were filled with phenol red free MEM, and then the corneas were immersed in the medium and equilibrated in an incubator set at 32°C ± 1 °C for 1 hr.

QUALITY CHECK OF THE ISOLATED CORNEAS:
Following the equilibration, the medium was collected and removed from both chambers of the corneal holders, and the chambers were filled with fresh phenol red free MEM. The baseline opacity value of each cornea was measured using the opacitometer (BASF-OP3.0, BASF).
The cornea was then macroscopically examined again for defects including opacity, scratches, and neovascularization. Corneas with abnormalities or opacity greater than 7 opacity units were not used in the study.

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE:
The test substance was rinsed off from the application side with phenol red-containing MEM for 3 times.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of UV/VIS spectrophotometry (OD490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS), IVIS = mean opacity value + (15x OD490 value)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as serious eye damage and labelled Category 1 according to CLP/EPS/GHS.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55 no prediction can be made.

Irritation parameter:

in vitro irritation score

Remarks:

mean value of 3 corneas

Run / experiment:

4 h

Value:

2.2

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Remarks:

negative control is a vehicle control

Positive controls validity:

valid

Other effects / acceptance of results:

The vehicle control (physiological saline) produced IVIS of -1.3, and was identified as non-irritant. While the positive control produced IVIS of 95.2 and was identified as a severe irritant or corrosive. The test substance produced mean IVIS of 2.2.

OTHER EFFECTS:
- Lesions and clinical observations: No abnormalities (scratches, neovascularization, etc.) were macroscopically observed in any cornea in the process of measurement.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The opacity and permeability values of the negative control were less than the upper limits (opacity, 2.4; permeability, 0.019) of the historical control opacity and permeability values.
- Acceptance criteria met for positive control: The IVIS value of the positive control was within 2 standard deviations (54.8 to 140.4) of the latest historical control mean.
Thus, the test results met the study acceptance criteria.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of the BCOP assay 20% solutions of the test substance in physiological saline was not irritating to the eye. Application of the test substance to bovine corneas resulted in a calculated mean IVIS of 2.2 (threshold for eye irritation IVIS > 3). Since 20% solution of the test substance was used, this study cannot be used for classification purpose.

Reference 5

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12 - 27 Feb 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

guideline was not yet released when study was conducted

GLP compliance:

yes (incl. QA statement)

Remarks:

Food and Consumer Product Safety Authority (VWA), Den Haag, the Netherlands

Species:

chicken

Strain:

other: ROSS, sping chickens

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: poultry slaughterhouse v.d. Bor, Nijkerkerveen, the Netherlands
- Number of animals:
- Characteristics of donor animals: males or females, 7 weeks old, body weight range approx. 2.0 - 3.0 kg
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they have reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
- Time interval prior to initiating testing: 2 h
- Indication of any existing defects or lesions in ocular tissue samples: Fluorescein-treated cornea were examined with a slit-lamp microscope to ensure that the cornea is not damaged.
- Indication of any antibiotics used: not specified

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 µL (5% solution) and 30 mg (neat test substance)
- Concentration: 5% aqueous solution and 100%

NEGATIVE CONTROL
- Amount applied: 30 µL
- Concentration: 0.9% physiological saline

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5% (w/w) aqueous solution

Duration of treatment / exposure:

10 sec

Duration of post- treatment incubation (in vitro):

240 min

Number of animals or in vitro replicates:

three test eyes per test concentration, negative and positive control

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and cornea were examined for damages with a slit-lamp microscope after fluorescein-treatment. If undamaged, the eye was further dissected from the head. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short. The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a rate of ca. 0.10 -0.15 mL/min. The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32 °C. After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they are not damaged. Corneal thickness was measured at the corneal apex of each eye (t0). Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes that show opacity (score higher than 0.5), or are unacceptably stained with fluorescein (score higher than 0.5) indicating the cornea to be permeable, or eyes that show any other signs of damage, were rejected as test eyes and replaced.

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

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: rinsing with 20 mL saline

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: slit-lamp microscope
- Damage to epithelium based on fluorescein retention: slit-lamp microscope
- Swelling: measured with optical pachymeter on a slit-lamp microscope
- Macroscopic morphological damage to the surface: slit-lamp microscope
- Others: After the final examination the test substance treated eyes and the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at 5 μM and stained with PAS (Periodic Acid-Schiff). Histopathological examination was performed by light microscopy.

SCORING SYSTEM:
- Mean corneal swelling (%): (corneal thickness at time t - corneal thickness at t0 / corneal thickness at t0) x 100
A negative swelling up to -5% (not unusual for control eyes) will be presented as 0% swelling.
The mean percentage of swelling for the three test eyes will be calculated for each of the observation time points of 30, 75, 120, 180, and 240 minutes.

- Mean maximum opacity score:
Opacity degree of density (area most dense taken for scoring)
0 = no opacity
0.5 = very faint opacity
1 = scattered or diffuse areas, details of iris clearly visible
2 = easily discernible translucent area, details of iris slightly obscured
3 = severe corneal opacity, no specific details of iris visible, size of pupil barely iscernible
4 = complete corneal opacity, iris invisible
The mean corneal opacity value for all test eyes is calculated for the observation time points of 30,
75, 120, 180, and 240 minutes. In case of score 4, the thickness assessment will not be possible.

- Mean fluorescein retention score at 30 minutes post-treatment:
0 = no fluorescein retention
0.5 = very minor single cell staining
1 = single cell staining scattered throughout the treated area of the cornea
2 = focal or confluent dense single cell staining
3 = confluent large areas of the cornea retaining tluorescein
If desired or in case of test substances that have adhered to the cornea, fluoresce in retention can be determined at t=240 min or whenever the test compound is removed.

DECISION CRITERIA:
- Mean corneal swelling (%):
-5 -5 --> Category I
6 - 12 --> Category II
13 -18 (> 75 min. after treatment) --> Category II
13 - 18 (< 75 min. after treatment) --> Category III
19 -26 (> 75 min. after treatment) --> Category III
27 - 32 (> 75 min. after treatment) --> Category III
27 - 32 (< 75 min. after treatment) --> Category IV
> 32 --> Category IV

- Mean max. opacity score:
0.0 -0.5 --> Category I
0.6 - 1.5 --> Category II
1.6 - 2.5 --> Category III
2.6 - 4.0 --> Category IV

- Mean fluorescein retention score at 30 minutes post-treatment:
0.0 -0.5 --> Category I
0.6 - 1.5 --> Category II
1.6 - 2.5 --> Category III
2.6 - 3.0 --> Category IV

Overall in vitro irritancy classifications:
Corrosive/Severe Irritant:
3 x IV
2 x IV, 1 x III
2 x IV, 1 x II*
2 x IV, 1 x I*
Corneal opacity ≥ 3 at 30 min (in at least 2 eyes)
Corneal opacity = 4 at any time point (in at least 2 eyes)
Severe loosening of the epithelium (in at least 1 eye)
* combination of categories less likely to occur.

In addition to the general irritancy class and regulatory classifications, an Irritation Index was given for the samples tested to allow for numerical ranking and comparison. The index was based on the addition of the maximum mean scores obtained for the parameters according to the following formula:
Irritation Index= maximum mean corneal swelling+ maximum mean opacity (x 20) + mean fluorescein score (x 20)
A factor of 20 was included to give equal weight to the scores obtained for opacity and fluorescein retention in the index compared to the maximum swelling possible (circa 60%). The maximum Irritation Index possible is ca. 200.

Irritation parameter:

percent corneal swelling

Run / experiment:

100%

Value:

7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Irritation Category II

Irritation parameter:

cornea opacity score

Remarks:

mean value of 3 eyes

Run / experiment:

100%

Value:

1.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Irritation Category II

Irritation parameter:

fluorescein retention score

Run / experiment:

100%

Value:

1.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Irritation Category II

Other effects / acceptance of results:

MICROSCOPIC EXAMINATION:
Microscopic examination of corneas after treatment with 5% solution or neat test substance revealed epithelial effects in both groups, i.e. very slight erosion and necrosis. No abnormalities were observed in the stroma or endothelium. Microscopic examination of the corneas generally confirmed the effects observed by slit-lamp examination.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control eyes did not show any significant corneal effect by slit-lamp and by microscopic observations and demonstrated that the general conditions during the test were adequate.
- Acceptance criteria met for positive control: The positive control eyes showed severe corneal effects by slit-lamp and by microscopic observations and demonstrated the suitability and sensitivity of the ICE to detect severe eye irritants.

Table 1 Results of slit-lamp examination.

	Maximum mean score			Irritation categories^1	Irritation index ^2
Treatment	Swelling %	Opacity	Fluorescein retention
Test substance 100%	7	1.5	1.5	II, II, II	67
Test substance 5%	6	1.2	1.2	II, II, II	54
Negative control (saline)	0	0.0	0.0	I, I, I	0
Positive control (5% BAC)	31*	3.0	3.0	III, IV, IV	151

^{BAC: benzalkonium chloride}

¹ l = no effect; II = slight effect; III = moderate effect; IV = severe effect

² Irritation Index= maximum mean corneal swelling+ maximum mean opacity (x 20) + mean fluorescein score (x 20)

* Blisters and severe loosing (detachment) of epithelium

 

                                                                                                  

Interpretation of results:

other: no prediction can be made

Conclusions:

The irritation potential of the test substance was assessed in the ICE assay. After treatment with the neat test substance corneal swelling, corneal opacity and fluorescein retention were all classed as category II, respectively. According to OECD 438 no prediction on the eye irritation potential can be made and additional testing should be conducted for classification and labeling purposes.

Reference 6

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

03 - 27 Feb 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

guideline was not yet released when study was conducted

GLP compliance:

yes (incl. QA statement)

Remarks:

Food and Consumer Product Safety Authority (VWA), Den Haag, the Netherlands

Species:

chicken

Strain:

other: ROSS, sping chickens

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: poultry slaughterhouse v.d. Bor, Nijkerkerveen, the Netherlands
- Number of animals:
- Characteristics of donor animals: males or females, 7 weeks old, body weight range approx. 2.0 - 3.0 kg
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they have reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
- Time interval prior to initiating testing: 2 h
- indication of any existing defects or lesions in ocular tissue samples: Fluorescein-treated cornea were examined with a slit-lamp microscope to ensure that the cornea is not damaged.
- Indication of any antibiotics used: not specified

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 µL (5% solution) and 30 mg (neat test substance)
- Concentration: 5% aqueous solution and 100%

NEGATIVE CONTROL
- Amount applied: 30 µL
- Concentration: 0.9% physiological saline

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5% (w/w) aqueous solution

Duration of treatment / exposure:

10 sec

Duration of post- treatment incubation (in vitro):

240 min

Number of animals or in vitro replicates:

three test eyes per test concentration, negative and positive control

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and cornea were examined for damages with a slit-lamp microscope after fluorescein-treatment. If undamaged, the eye was further dissected from the head. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short. The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a rate of ca. 0.10 -0.15 mL/min. The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32 °C. After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they are not damaged. Corneal thickness was measured at the corneal apex of each eye (t0). Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes that show opacity (score higher than 0.5), or are unacceptably stained with fluorescein (score higher than 0.5) indicating the cornea to be permeable, or eyes that show any other signs of damage, were rejected as test eyes and replaced.

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

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: rinsing with 20 mL saline

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: slit-lamp microscope
- Damage to epithelium based on fluorescein retention: slit-lamp microscope
- Swelling: measured with optical pachymeter on a slit-lamp microscope
- Macroscopic morphological damage to the surface: slit-lamp microscope
- Others: After the final examination the test substance treated eyes and the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at 5 μM and stained with PAS (Periodic Acid-Schiff). Histopathological examination was performed by light microscopy.

SCORING SYSTEM:
- Mean corneal swelling (%): (corneal thickness at time t - corneal thickness at t0 / corneal thickness at t0) x 100
A negative swelling up to -5% (not unusual for control eyes) will be presented as 0% swelling.
The mean percentage of swelling for the three test eyes will be calculated for each of the observation time points of 30, 75, 120, 180, and 240 minutes.

- Mean maximum opacity score:
Opacity degree of density (area most dense taken for scoring)
0 = no opacity
0.5 = very faint opacity
! = scattered or diffuse areas, details of iris clearly visible
2 = easily discernible translucent area, details of iris slightly obscured
3 = severe corneal opacity, no specific details of iris visible, size of pupil barely iscernible
4 = complete corneal opacity, iris invisible
The mean corneal opacity value for all test eyes is calculated for the observation time points of 30,
75, 120, 180, and 240 minutes. In case of score 4, the thickness assessment will not be possible.

- Mean fluorescein retention score at 30 minutes post-treatment:
0 = no fluorescein retention
0.5 = very minor single cell staining
1 = single cell staining scattered throughout the treated area of the cornea
2 = focal or confluent dense single cell staining
3 = confluent large areas of the cornea retaining tluorescein
If desired or in case of test substances that have adhered to the cornea, fluoresce in retention can be determined at t=240 min or whenever the test compound is removed.

DECISION CRITERIA:
- Mean corneal swelling (%):
-5 -5 --> Category I
6 - 12 --> Category II
13 -18 (> 75 min. after treatment) --> Category II
13 - 18 (< 75 min. after treatment) --> Category III
19 -26 (> 75 min. after treatment) --> Category III
27 - 32 (> 75 min. after treatment) --> Category III
27 - 32 (< 75 min. after treatment) --> Category IV
> 32 --> Category IV

- Mean max. opacity score:
0.0 -0.5 --> Category I
0.6 - 1.5 --> Category II
1.6 - 2.5 --> Category III
2.6 - 4.0 --> Category IV

- Mean fluorescein retention score at 30 minutes post-treatment:
0.0 -0.5 --> Category I
0.6 - 1.5 --> Category II
1.6 - 2.5 --> Category III
2.6 - 3.0 --> Category IV

Overall in vitro irritancy classifications:
Corrosive/Severe Irritant:
3 x IV
2 x IV, 1 x III
2 x IV, 1 x II*
2 x IV, 1 x I*
Corneal opacity ≥ 3 at 30 min (in at least 2 eyes)
Corneal opacity = 4 at any time point (in at least 2 eyes)
Severe loosening of the epithelium (in at least 1 eye)
* combination of categories less likely to occur.

In addition to the general irritancy class and regulatory classifications, an Irritation Index was given for the samples tested to allow for numerical ranking and comparison. The index was based on the addition of the maximum mean scores obtained for the parameters according to the following formula:
Irritation Index= maximum mean corneal swelling+ maximum mean opacity (x 20) + mean fluorescein score (x 20)
A factor of 20 was included to give equal weight to the scores obtained for opacity and fluorescein retention in the index compared to the maximum swelling possible (circa 60%). The maximum Irritation Index possible is ca. 200.

Irritation parameter:

percent corneal swelling

Run / experiment:

100%

Value:

11

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Irritation Category II

Irritation parameter:

cornea opacity score

Remarks:

mean value of 3 eyes

Run / experiment:

100%

Value:

1.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Irritation Category III

Irritation parameter:

fluorescein retention score

Run / experiment:

100%

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Irritation Category III

Other effects / acceptance of results:

MICROSCOPIC EXAMINATION:
Microscopic examination of corneas after treatment with 5% solution or neat test substance revealed epithelial effects in both groups, 5% solution: very slight or slight erosion and very slight or slight vacuolation; neat substance: slight or moderate erosion, necrosis and slight or moderate vacuolation. In addition to these diffuse observed effects, focal spot(s) with severe vacuolation extending into deeper epithelial layers were observed, causing a dent. No abnormalities were observed in the stroma or endothelium for both concentrations. Microscopic examination of the corneas generally confirmed the effects observed by slit-lamp examination.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control eyes did not show any significant corneal effect by slit-lamp and by microscopic observations and demonstrated that the general conditions during the test were adequate.
- Acceptance criteria met for positive control: The positive control eyes showed severe corneal effects by slit-lamp and by microscopic observations and demonstrated the suitability and sensitivity of the ICE to detect severe eye irritants.

Table 1 Results of slit-lamp examination.

	Maximum mean score			Irritation categories^1	Irritation index ^2
Treatment	Swelling %	Opacity	Fluorescein retention
Test substance 100%	11*	1.7	2.0	II, III, III	85
Test substance 5%	10	0.5	1.0	II, I, II	40
Negative control (saline)	0	0.0	0.0	I, I, I	0
Positive control (5% BAC)	33**	3.0	3.0	IV, IV, IV	153

^{BAC: benzalkonium chloride}

¹ l = no effect; II = slight effect; III = moderate effect; IV = severe effect

² Irritation Index= maximum mean corneal swelling+ maximum mean opacity (x 20) + mean fluorescein score (x 20)

* Slight erosion of epithelium

** Blisters and severe loosening (detachment) of epithelium

 

                                                                                                  

Interpretation of results:

other: no prediction can be made

Conclusions:

The irritation potential of the test substance was assessed in the ICE assay. After treatment with the neat test substance corneal swelling, corneal opacity and fluorescein retention were classed as category II,III and III, respectively. According to OECD 438 no prediction on the eye irritation potential can be made and additional testing should be conducted for classification and labeling purposes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin

In vivo

The skin irritation potential of the test substance was determined by an in vivo skin irritation test (Richeux, 2006a) in albino rabbits according to OECD Guideline 404 and in compliance with GLP. An amount of 0.5 g of the test substance was applied to the intact skin of three rabbits under semi-occlusive conditions for 4 hours. After the exposure period the patch was removed and scores were taken 1, 24, 48 and 72 hours after patch removal using the Draize scoring system. No erythema and oedema were observed at any observation time point. Mean scores at 24/48/72 hours for each animal were 0.0, 0.0 and 0.0 for both, erythema and oedema. Based on the results, the undiluted test substance was not irritating to the skin under the conditions of the test.

The skin irritation potential of the test substance was determined by an in vivo skin irritation test (Richeux, 2006b) in albino rabbits according to OECD Guideline 404 and in compliance with GLP. An amount of 0.5 g of the test substance was applied to the intact skin of three rabbits under semi-occlusive conditions for 4 hours. After the exposure period the patch was removed and scores were taken 1, 24, 48 and 72 hours after patch removal using the Draize scoring system. No erythema and oedema were observed at any observation time point. Mean scores at 24/48/72 hours for each animal were 0.0, 0.0 and 0.0 for both, erythema and oedema. Based on the results, the undiluted test substance was not irritating to the skin under the conditions of the test.

The skin irritation potential of the test substance was determined by a supporting in vivo skin irritation test (Kreiling, 1996) in albino rabbits according to OECD Guideline 404 and in compliance with GLP. An amount of 0.5 g of the test substance pasted with 0.5 mL physiological saline was applied to the skin of three rabbits under semi-occlusive conditions for 4 hours. After the exposure period the patch was removed and residual test material was removed by warm tap water. Scores were taken 30-60 min, 24, 48 and 72 hours after patch removal using the Draize scoring system. 30 - 60 min after patch removal animals showed very slight to well-defined erythema (score 1-2), in two animals reversible within 24 h and in one animal reversible within 48 h. No edema was observed in any animal. Mean scores for erythema at 24/48/72 hours for each animal were 0.3, 0.0 and 0.0, respectively and for edema it was 0.0 for all 3 animals. Based on the results, the undiluted test substance was not irritating to the skin under the conditions of the test.

 

Eye

In vivo

The eye irritation potential of the test substance was investigated in three albino rabbits according to OECD Guideline 405 (Kreiling, 1996) and in compliance with GLP. An amount of 0.1 g of the test substance was placed in one eye of each animal. The other eye remained untreated and served as control. The eyes were examined and scored according to the Draize scoring system 1, 24, 48 and 72 hours and 7 days after instillation. Corneal opacity (score 1-2) was observed in all animals 24 h after instillation reversible within 2 or 7 days, respectively. Redness of the iris (score 1) was observed in two animals 1 hour or one day after instillation of the test substance reversible within 7 or 2 days, respectively. A redness and swelling of the conjunctivae (both score 3) of the treated eyes was observed in all three animals on the day of instillation reversible within 7 days. The mean scores at 24/48/72 hour for each animal were 1.0, 0.3 and 1.3 for cornea, 1.0, 0.3 and 0.0 for iris, 2.7, 2.3 and 2.7 for redness and 1.0,1.0 and 1.0 for chemosis of conjunctivae, respectively. Based on the results of this study, the test substance was irritating to the eyes under the conditions of the test.

The eye irritation potential of the test substance was investigated in three albino rabbits according to OECD Guideline 405 (Busschers, 1997) and in compliance with GLP. An amount of 88 mg of the test substance was placed in one eye of each animal. The other eye remained untreated and served as control. The eyes were examined and scored according to the Draize scoring system 1, 24, 48, 72 hours, 7 and 14 days after instillation. Corneal opacity (score 1) was observed in one animal 24 - 72 hour after instillation reversible within 7 days. Redness of the iris (score 1) was observed in all animals 1 hour after instillation reversible within 24, 48 and 72 hour, respectively. Redness of the conjunctivae (score 1-3) was observed in all animals 1 h after instillation of the test substance reversible within 7 or 14 days. A swelling of the conjunctivae (score 4) was observed in all three animals 1 hour after instillation reversible within 72 hours or 7 days. In addition, reduced elasticity of the eyelids was observed in two animals 48 and 72 hours after instillation. The mean scores at 24/48/72 h for each animal were 0,0 and 1.0 for cornea, 0.3, 0 and 0.7 for iris, 2.0, 2.0 and 2.7 for redness and 1.3, 1.3 and 1.7 for chemosis of conjunctivae, respectively. Based on the results of this study, the test substance was irritating to the eyes under the conditions of the test.

In vitro

The eye irritation potential of the test substance was determined in a bovine corneal opacity and permeability test (BCOP test) according to OECD Guideline 437 and in compliance with GLP (Furukawa, 2017). After a first opacity measurement of the fresh bovine corneas, 0.75 mL of a 20% (w/v) solution of the test substance in saline was applied directly to the epithelial surface of three cattle corneas in an incubation chamber in horizontal position for 4 hours at 32 ± 1 °C. After the incubation phase the test substance was rinsed from the corneas. Afterwards, opacity was measured a second time. In addition, the permeability of the corneas was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 min at 32 ± 1 °C. The results of the opacity and permeability measurement of the test substance were used to calculate an in vitro irritation score (IVIS) of 13.8. With the negative control saline neither an increase of opacity nor permeability of the corneas could be observed. The mean IVIS of the negative control was 0.1. The mean IVIS of the positive control (imidazole 20% (w/v)) was 100.4, thus confirming the validity of the test system. Based on the results, the test substance was non-corrosive to the eye under the conditions of the test. According to OECD Guideline 437, IVIS for the test substance is in the range of >3 to <=55. Therefore, no prediction on the irritation potential of the test substance can be made.

Further, the eye irritation potential of the test substance was determined in a bovine corneal opacity and permeability test (BCOP test) according to OECD Guideline 437 and in compliance with GLP (Sakakibara, 2016). After a first opacity measurement of the fresh bovine corneas, 0.75 mL of a 20% (w/v) solution of the test substance in saline was applied directly to the epithelial surface of three cattle corneas in an incubation chamber in horizontal position for 4 hours at 32 ± 1 °C. After the incubation phase the test substance was rinsed from the corneas. Afterwards, opacity was measured a second time. In addition, the permeability of the corneas was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 min at 32 ± 1 °C. The results of the opacity and permeability measurement of the test substance were used to calculate an in vitro irritation score (IVIS) of 2.2. With the negative control saline neither an increase of opacity nor permeability of the corneas could be observed. The mean IVIS of the negative control was -1.3. The mean IVIS of the positive control (imidazole 20% (w/v)) was 95.2, thus confirming the validity of the test system. Based on the results, the test substance was not irritating to the eye. Since 20% solution of the test substance was used, this study cannot be used for classification purpose.

The eye irritation potential of the test substance was further determined in an isolated chicken eye test (ICE) according to OECD Guideline 438 and in compliance with GLP. In two independent studies (Prinsen, 2009a and 2009b), three isolated chicken eyes per group were exposed to a single application of the test substance at 5% (w/v) in aqueous solution or the neat substance for 10 sec using an application volume of 30 μL or 30 mg (solid). An additional control group was treated with physiological saline. Three main parameters were measured by slit-lamp microscope: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. In addition, histopathology was performed on the cornea to assess the nature and depth of injury.

In the first study (Prinsen, 2009a), theneat test substancecaused slight swelling (7%), slight to moderate opacity (1.5) and slight to moderate fluorescein retention (1.5). The calculated Irritation Index was 67. The5% solutioncaused slight swelling (6%), slight or slight to moderate opacity (1.2) and slight or slight to moderate fluorescein retention (1.2). The negative control (physiological saline) caused no corneal effects. The calculated Irritation Index was 0. The positive control benzalkonium chloride 5% caused moderate severe swelling (31%), severe opacity (3.0) and severe fluorescein retention (3.0). The calculated Irritation Index was 151. Microscopic examination of the corneas generally confirmed the effects observed by slit-lamp examination.

In the second study (Prinsen, 2009b), theneat test substancecaused overall slight opacity and slight fluorescein retention and several focal areas with moderate corneal opacity (1.7) and moderate fluorescein retention (2.0). In addition, slight swelling (11 %) and slight erosion of the epithelium were observed. The calculated Irritation Index was 85. The 5% solution caused slight swelling (10%), very slight opacity (0.5) and slight fluorescein retention (1.0). The calculated Irritation Index was 40. The negative control (physiological saline) caused no corneal effects. The calculated Irritation Index was 0. The positive control benzalkonium chloride 5% caused moderate severe swelling (31%), severe opacity (3.0) and severe fluorescein retention (3.0). The calculated Irritation Index was 151. Microscopic examination of the corneas generally confirmed the effects observed by slit-lamp examination.

Based on the results, the test substance was non-corrosive to the eye under the conditions of both chicken eye studies but no prediction on the irritation potential can be made.

 

For eye irritation a weight of evidence approach based on 2 in vivo studies and 4 ex vivo studies was conducted. Both in vivo studies in rabbits showed an irritating potential of the test substance, while one ex vivo study showed no irritation on bovine cornea (Sakakibara, 2016) and 3 further ex vivo studies in bovine cornea (Furukawa, 2017) and isolated chicken eyes (Prinsen, 2009a and 2009b) for which based on results no prediction can be made regarding classification purposes. In conclusion, following the weight of evidence approach, the test substance was regarded as irritating to the eye, based on in vivo studies.

Justification for classification or non-classification

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

The available data on eye irritation meet the criteria for classification as Eye Irrit. 2 (H319) according to Regulation (EC) 1272/2008.