rac-(1R,4S,4aR,8R,8aS)-9-(dichloromethylidene)-8-hydroxyoctahydro-1,4-methanonaphthalen-5(1H)-one

Endpoint summary

Description of key information

- Skin irritation/corrosion, not irritating, in vivo, male, rabbit, OECD 404, Zelenák 2014

- Eye irritation/corrosion, not irritating, ex vivo, chicken, OECD 438, Hargitai 2014

- Eye irritation/corrosion, not irritating, in vivo, male, rabbit, OECD 405, Zelenák 2014

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Remarks:

This study was conducted solely to comply with a non-EU national registration requirement.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Mar 2014 to 16 Mar 2014

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 April 24, 2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 12 weeks
- Weight at study initiation: 2979 – 3262 g
- Housing: Animals were housed individually in AAALAC approved metal wire rabbit cages. Cages are of an open wire structure and cages are placed together to allow some social interaction with rabbit(s) in adjoining cages.
- Diet: UNI diet for rabbits, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 26 Feb to 10/12 Mar 2014

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 24-48
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
11 Mar 2014 to 16 Mar 2014

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Remarks:

(moistened with a small amount of water)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 g of the test substance (moistened with small amount of water)

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: back and flanks
- % coverage: 2.5 x 2.5 cm
- Type of wrap if used: This gauze pad was applied to the intact skin of the clipped area and was kept in contact with the skin by a patch with a surrounding adhesive hypoallergenic plaster. The entire trunk of the animals was then wrapped with plastic wrap held in place with an elastic stocking.

REMOVAL OF TEST SUBSTANCE
- Washing: The dressing was removed and the skin was flushed with lukewarm tap water to clean the application site.
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
- Clinical signs, including viability/mortality, were recorded daily from the day of application of the animals to the termination of the test.
- Body weights were recorded on the day of application and the end of the experiment.

SCORING SYSTEM:
- Method of calculation: Draize scoring system. The skin reaction was assessed at approximately 1, 24, 48 and 72 hours after the end of exposure (removal of the dressing, gauze patch and test item). The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for oedema grades, separately. The Cumulative Scores for the Skin Irritation Scores were calculated and represent the sum of all numerical scores for each animal at each time point. The resulting Mean Cumulative Skin Irritation Score was calculated for all animals at each time point.
The Primary Irritation Index (P.I.I.) was calculated by totalling the mean cumulative scores at 24, 48 and 72 hours and then dividing by the number of data points. Skin irritation was scored based on the grading system presented in 'Any other information on materials methods incl. tables'.

Irritation parameter:

erythema score

Basis:

animal: #1, #2, #3

Time point:

24/48/72 h

Score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal: #1, #2, #3

Time point:

24/48/72 h

Score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

other: #1, #2, #3

Time point:

24/48/72 h

Score:

0

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

The primary irritation index was 0.00 (out of a maximum score of 8.0). No corrosive effects were noted on the treated skin of any animal at any of the observation intervals.
No local dermal signs were observed in the treated animals throughout the study.

Other effects:

No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.
The body weights of all rabbits were considered to be within the normal range of variability.

Table 1. Skin Irritation Scores - Mean Values After 24, 48 and 72 Hours

Animal Number	Sex	Erythema	N	Oedema	N	Primary Skin Irritation Index
00840	male	0.00	3	0.00	3	0.00
00870	male	0.00	3	0.00	3
00842	male	0.00	3	0.00	3
Mean score		0.00		0.00
N= number of available data points.

Interpretation of results:

GHS criteria not met

Conclusions:

The application of the test substance did not result in any signs of skin irritation. According to the Draize classification criteria, the test substance is considered to be “not irritant” to rabbit skin.

Executive summary:

In this GLP compliant, OECD 404 study, the primary skin irritation potential of the test substance was investigated using 3 young adult New Zealand White rabbits. The animals were treated by topical, semi-occlusive application of 0.5 g to their intact shaved flanks. The duration of treatment was 4 hours. The scoring of skin reactions was performed at 1, 24, 48 and 72 hours after removal of the dressing. The primary irritation index (P.I.I.) was calculated by totalling the mean cumulative scores at 24, 48 and 72 hours and then dividing by the number of data points.

The primary irritation index was 0.00. No local dermal signs were observed in the treated animals throughout the study. No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred. As no clinical signs were observed up to 72 hours after patch removal, the study was terminated after the 72 hour observation. The body weights of all rabbits were considered to be within the normal range of variability.

The application of the test substance did not result in any signs of skin irritation. According to the Draize classification criteria, the test substance is considered to be “not irritant” to rabbit skin (P.I.I. = 0.00).

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:

27 May 2014 to 01 Jun 2014

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: 2 October 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 14 weeks
- Weight at study initiation: 3390 g – 3588 g
- Housing: Rabbits were individually housed in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages.
- Diet: UNI diet for rabbit, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 27 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 30 - 80
- Air changes (per hr): 15 – 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
27 May 2014 to 01 Jun 2014

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount applied: A single dose of 0.1 g of test item was administered to the left eye of each animal (n=3).

Duration of treatment / exposure:

One hour

Observation period (in vivo):

Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours in all animals after test material installation. Observations with fluorescein staining were made approximately 24 hours before treatment and then 24, 48 and 72 hours after treatment in all animals.
The duration of the observation period was sufficient to identify reversibility or irreversibility of changes. Any clinical signs of toxicity or signs of ill-health during the study were recorded. All rabbits were examined for distress at least twice daily, with observations at least 6 hours apart. Clinical observations or signs of ill-health were recorded.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: The treated eye was rinsed with physiological saline solution at the first observation time point at one hour after the application of test item on all animals as residual test item was in the eye.
- Time after start of exposure: One hour

SCORING SYSTEM:
The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (2nd October 2012). See ‘Any other information on materials and methods incl. tables’.

TOOL USED TO ASSESS SCORE: Observations with fluorescein staining were made approximately 24 hours before treatment and then 24, 48 and 72 hours after treatment in all animals.

Irritation parameter:

overall irritation score

Basis:

animal: #1, #2, #3

Time point:

24/48/72 h

Score:

0

Remarks on result:

other: Weak irritation observed at the 1 hour reading (conjunctivae), fully reversible within 24 hours.

Irritation parameter:

cornea opacity score

Basis:

animal: #1, #2, #3

Time point:

24/48/72 h

Score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal: #1, #2, #3

Time point:

24/48/72 h

Score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal: #1, #2, #3

Time point:

24/48/72 h

Score:

0

Remarks on result:

other: Weak irritation observed at the 1 hour reading, fully reversible within 24 hours.

Irritation parameter:

chemosis score

Basis:

animal: #1, #2, #3

Time point:

24/48/72 h

Score:

0

Remarks on result:

other: Weak irritation observed at the 1 hour reading, fully reversible within 24 hours.

Irritant / corrosive response data:

Initial Pain Reaction/Pain reaction (IPR/PR) was not observed. Conjunctival redness (score 1), chemosis (score 1) and discharge (score 1) were seen in all rabbits at 1 hour after treatment. Residual test item was noted on the eye in all animals at 1 hour after treatment. No other signs were observed and all animals were symptom free 24 hours after treatment. Fluorescein staining was negative in all animals at all-time points during the study.
The control eyes were symptom-free during the study.
Individual ocular reactions and individual total sores results are presented in Table 1 and 2 in 'Any other information on results incl. tables'.

Other effects:

The body weights of all rabbits were considered to be within the normal range of variability.
No clinical signs of systemic toxicity were observed in any animals in this study.
No mortality occurred in this study.

Table 1. Individual Draize Scores and Individual Total Scores* for Ocular Irritation

Rabbit number and sex	#1 Male				#2 Male				#3 Male
	PR/PR = 0				IPR/PR = 0				IPR/PR = 0
Time after treatment	1 Hr	24 Hr	48 Hr	72 Hr	1 Hr	24 Hr	48 Hr	72 Hr	1 Hr	24 Hr	48 Hr	72 Hr
CORNEA
E = Degree of Opacity	0	0	0	0	0	0	0	0	0	0	0	0
F = Area of Cornea	0	0	0	0	0	0	0	0	0	0	0	0
involved
*Score (E x F) x 5	0	0	0	0	0	0	0	0	0	0	0	0
IRIS D	0	0	0	0	0	0	0	0	0	0	0	0
*Score (D x 5)	0	0	0	0	0	0	0	0	0	0	0	0
CONJUNCTIVAE
A = Redness	1	0	0	0	1	0	0	0	1	0	0	0
B = Chemosis	1	0	0	0	1	0	0	0	1	0	0	0
C = Discharge	1	0	0	0	1	0	0	0	1	0	0	0
*Score (A+B+C) x 2	6	0	0	0	6	0	0	0	6	0	0	0
*Total Score	6	0	0	0	6	0	0	0	6	0	0	0

IPR: Initial pain reaction Hr: Hour(s)

PR: Pain reaction

Table 2. Individual Total Scores and Group Mean Scores for Ocular Irritation Calculated from the Draize Scores

Rabbit Number and Sex	*Individual Total Scores At:
	1 Hour	24 Hours	48 Hours	72 Hours
01021 Male	6	0	0	0
01038 Male	6	0	0	0
01043 Male	6	0	0	0
Group Total	18	0	0	0
Group Mean Score	6	0	0	0

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was graded as a minimal irritant (Class 3 on a 1 to 8 scale) to the rabbit eye according to the modified Kay and Calandra classification system.

Executive summary:

In this GLP compliant OECD 405 study, the primary eye irritation effect of the test item was investigated using 3 young adult male New Zealand White rabbits. The test item was administered as an installation of a single dose of 0.1 g into the conjunctival sac of the left eye with the untreated right eyes serving as the control. The scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours in all animals after test material instillation. Observations with fluorescein staining were made approximately 24 hours before treatment and then 24, 48 and 72 hours after treatment in all animals. Rabbits were treated with analgesic and anaesthetic as per the regulatory guideline. Three animals were used to make the classification.

Initial Pain Reaction/Pain reaction (IPR/PR) was not observed. Conjunctival redness (score 1), chemosis (score 1) and discharge (score 1) were seen in all rabbits at 1 hour after treatment. Residual test item was noted on the eye in all animals at 1 hour after treatment. No other signs were observed and all animals were symptom free 24 hours after treatment. Fluorescein staining was negative in all animals at all-time points during the study. The control eyes were symptom-free during the study. No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred. The body weights of all rabbits were considered to be within the normal range of variability.

The test item was graded as a minimal irritant (Class 3 on a 1 to 8 scale) to the rabbit eye according to the modified Kay and Calandra classification system.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 Apr 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26 July 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

9 December 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol 80: Chicken Enucleated Eye Test (CEET)

Version / remarks:

1994

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Local abattoir
- Characteristics of donor animals (e.g. age, sex, weight): young chickens, approximately 7 weeks old.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with physiological saline solution, then placed in a lockable plastic box (4-5 heads per box). The heads were immediately transported at ambient temperature.
- Time interval prior to initiating testing: The heads were received and processed at the Test Facility within approximately 2 hours after collection.
- indication of any existing defects or lesions in ocular tissue samples: only eyes with appropriate quality were selected.
- Indication of any antibiotics used: Not examined

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Duration of treatment / exposure:

10 seconds

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids were carefully cut away with scissors, avoiding damaging the cornea. Cornea integrity was checked by applying one small drop of 2% (w/v) fluorescein solution onto the cornea surface for a few seconds and then subsequently rinsed off with 20 mL physiological saline solution. The fluorescein-treated cornea was examined with a hand-held slit lamp, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.
The eye ball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box (thus the appropriate humidity was maintained).
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head), again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was 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 physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minutes or 0.1-0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than +5% or -7% between the start of equilibration (-45 min) and the zero time. Slight change in thickness (1.4%) was observed in the eyes of this study, but this fact was considered normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured.

NUMBER OF REPLICATES
Three test item treated eyes, three positive control treated eyes and one negative control eye were examined during the study.

SOLVENT CONTROL USED: physiological saline, exposure duration was 10 seconds.

POSITIVE CONTROL USED: 30 mg powdered Imidazole, exposure duration was 10 seconds.

APPLICATION DOSE AND EXPOSURE TIME: 30 mg of test item, exposure duration was 10 seconds.

OBSERVATION PERIOD
- The control eyes and test item treated eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Slit-lamp microscope was used for the measurements.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test item or positive control substance if possible.
In case of solid material was stuck on the surface of the cornea after treatment and/or at later observation point(s), an additional gentle rinsing with 20 mL saline was performed and the rate of saline-drops was increased at each observation time point.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was scored using the area of the cornea that was most densely opacified. The change is corneal opacity was calculated for each eye at each timepoint. An overall maximum opacity score was calculated at 30 to 240 minutes minus the baseline cornea opacity of the individual eye.
- Damage to epithelium based on fluorescein retention. The change in fluorescein retention was calculated for each eye by correcting for the baseline reading. An overall fluorescein retention was calculated for all eyes at the 30 minutes reading.
- Swelling: Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope. The change in corneal thickness was calculated for each eye at 75 and 240 minutes relatively to the baseline reading. For each timepoint an overall average was calculated.

SCORING SYSTEM:
See ‘Any other information on materials and methods incl. tables’.

DECISION CRITERIA:
See ‘Any other information on materials and methods incl. tables’.

Irritation parameter:

percent corneal swelling

Run / experiment:

#1, #2

Value:

2.9

Vehicle controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

#3

Value:

0

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

#1, #2, #3

Value:

0

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein retention score

Run / experiment:

#1, #2, #3

Value:

0

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The results from all eyes used met the quality control standards. The negative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Valid. See Table 2 in ‘Any other information on results incl. tables’.
- Acceptance criteria met for positive control: Valid. See Table 3 in ‘Any other information on results incl. tables’.
- Range of historical values if different from the ones specified in the test guideline: See table 2 and 3 in ‘Any other information on results incl. tables’.

Table 1. Results test item

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	1.9%	I
Mean maximum corneal opacity change	0.0	I
Mean fluorescein retention change	0.0	I
Other Observations	Remaining test item on the surface of the cornea was detected up to the 240-min observation point (2 out of 3) or until the 120- min observation point (1 out of 3)
Overall ICE Class	3 x I

 

Table 2. Negative Control data including historical control data (physiological saline solution, 0.9% (w/v) NaCl)

Observation	Test Results	ICE Class	Historical Minimum value	Historical Maximum value
Maximum corneal swelling at up to 75 min	1.4%	I	0.0%	1.2%
Maximum corneal swelling at up to 240 min	1.4%	I	0.0%	1.2%
Maximum corneal opacity change	0.00	I	0.00	0.00
Fluorescein retention	0.00	I	0.00	0.00
Overall ICE Class	3 x I

Historical control data (n=17, data from 2013)

 

Table 3. Positive Control data including historical control data (Imidazole)

Observation	Test Results	ICE Class	Historical Minimum value	Historical Maximum value
Maximum corneal swelling at up to 75 min	2.4%	I	1.1%	5.9%
Maximum corneal swelling at up to 240 min	9.0%	II	4.6%	10.6%
Maximum corneal opacity change	4.00	IV	3.50	4.00
Fluorescein retention	3.00	IV	2.00	3.00
Overall ICE Class	1 x II and 2 x IV

Historical control data (n=17, data from 2013)

Interpretation of results:

GHS criteria not met

Remarks:

An in vivo study is required to conclude on classification.

Conclusions:

Based on this in vitro eye irritation test in isolated chicken eyes, the test item is not classified as severe irritant and not classified as non-irritant. It is concluded that an in vivo study is required for classification.

Executive summary:

In this GLP compliant OECD 438 study, the eye irritation potential of the test substance was evaluated in vitro in isolated chicken’s eyes. After the time zero reference measurements, the eyes were held in a horizontal position and the treatments were applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds, the surface of the eyes was rinsed with isotonic saline solution. Three eyes were treated with 30 mg of test item. Three positive control eyes were treated in a similar way with 30 mg of Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9% NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) was evaluated.

Minor corneal swelling (mean maximum value of 1.9%) was observed during the four hours observation period in the test item treated eyes. The test item remained adhered to the corneas. No other corneal effect was observed. The results from all eyes used in the study met the quality control standards. The negative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid.

Based on this in vitro eye irritation test in isolated chicken eyes, the test item is not classified as severe irritant and not classified as non-irritant. It is concluded that an in vivo study is required for classification.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vivo skin irritation/corrosion

In a GLP compliant, OECD 404 study, the primary skin irritation potential the test substance was investigated using 3 young adult New Zealand White rabbits (Zelenák, 2014). The animals were treated by topical, semi-occlusive application of 0.5 g to their intact shaved flanks. The duration of treatment was 4 hours. The scoring of skin reactions was performed at 1, 24, 48 and 72 hours after removal of the dressing. The primary irritation index (P.I.I.) was calculated by totalling the mean cumulative scores at 24, 48 and 72 hours and then dividing by the number of data points.

The primary irritation index was 0.00. No local dermal signs were observed in the treated animals throughout the study. No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred. As no clinical signs were observed up to 72 hours after patch removal, the study was terminated after the 72 hour observation. The body weights of all rabbits were considered to be within the normal range of variability.

The application of the test substance did not result in any signs of skin irritation. According to the Draize classification criteria, the test substance is considered to be “not irritant” to rabbit skin.

In vivo eye irritation/corrosion

In a GLP compliant OECD 405 study, the primary eye irritation effect of the test item was investigated using 3 young adult male New Zealand White rabbits (Zelenák, 2014). The test item was administered as an installation of a single dose of 0.1 g into the conjunctival sac of the left eye with the untreated right eyes serving as the control. The scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours in all animals after test material instillation. Observations with fluorescein staining were made approximately 24 hours before treatment and then 24, 48 and 72 hours after treatment in all animals. Rabbits were treated with analgesic and anaesthetic as per the regulatory guideline. Three animals were used to make the classification.

Initial Pain Reaction/Pain reaction (IPR/PR) was not observed. Conjunctival redness (score 1), chemosis (score 1) and discharge (score 1) were seen in all rabbits at 1 hour after treatment. Residual test item was noted on the eye in all animals at 1 hour after treatment. No other signs were observed and all animals were symptom free 24 hours after treatment. Fluorescein staining was negative in all animals at all-time points during the study. The control eyes were symptom-free during the study. No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred. The body weights of all rabbits were considered to be within the normal range of variability.

The test item was graded as a minimal irritant (Class 3 on a 1 to 8 scale) to the rabbit eye according to the modified Kay and Calandra classification system.

Ex vivo eye irritation/corrosion

In a GLP compliant OECD 438 study, the eye irritation potential of the test substance was evaluated in vitro in isolated chicken’s eyes (Hargitai, 2014). After the time zero reference measurements, the eyes were held in a horizontal position and the treatments were applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds, the surface of the eyes was rinsed with isotonic saline solution. Three eyes were treated with 30 mg of test item. Three positive control eyes were treated in a similar way with 30 mg of Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9% NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) was evaluated.

Minor corneal swelling (mean maximum value of 1.9%) was observed during the four hours observation period in the test item treated eyes. The test item remained adhered to the corneas. No other corneal effect was observed. The results from all eyes used in the study met the quality control standards. The negative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid.

Based on this in vitro eye irritation test in isolated chicken eyes, the test item is not classified as severe irritant and not classified as non-irritant. It is concluded that an in vivo study is required for classification.

Justification for classification or non-classification

Based on the results of GLP-compliant guideline studies on skin and eye irritation, classification is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.