Pentanediamide, N1,N5-dibutyl-2-[(2-ethyl-1-oxohexyl)amino]-, (2S)-

Endpoint summary

Description of key information

A skin irritation study according to EU Test Method B.4 was conducted on the test material. The test item did not elicit any skin reactions at the application site of any animal at any of the observation times (all scores 0). The mean values from 24 to 72 hours were therefore 0 for erythema and 0 for oedema. The test item caused no staining of the treated skin. No corrosive effects were noted on the treated skin of any animal at any measuring interval.

An in vivo eye irritation study according to EU Test Method B.5 was conducted on the test material. The primary irritation score was calculated by totaling the individual cumulative scores at 24, 48 and 72 hours and then dividing the resulting total by the number of data points. The primary irritation score was 0.56 (max. 13). The eye reactions (mean values from 24 to 72 hours) consisted of grade 0.00 corneal opacity, grade 0.00 iris lesions, grade 0.56 redness of the conjunctivae and grade 0.00 chemosis of the conjunctivae. No abnormal findings were observed in the cornea or the iris in any animal at any reading. No staining of the treated eyes by the test item was observed. No corrosion was observed at any of the measuring intervals. All eye reactions had cleared within 7 days after treatment.

An in vitro eye irritation study is also provided as a supporting study and indicates that the substance should not be classified as a severe eye irritant.

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:

27 February 2001 - 9 March 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

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:
Source: Elevage Scientifique des Dombes
F-01400 Chatillon sur Chalaronne, France
Number of animals: 1 male and 2 females
Age at start of treatment: 11-12 weeks (male) 9-11 weeks (females)
Body weights at start of treatment : 2.3-2.5 kg
Identification: By unique cage number and corresponding ear number.
Acclimatization: Under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study.
Allocation: Male No. 7 Female Nos. 8 and 9

Husbandry:
Room no.: 106 / RCC Ltd, Füllinsdorf
Conditions: Standard Laboratory Conditions
Air-conditioned with target ranges for room temperature 20 ± 3 °C, relative humidity 30-70 % and approximately 10-14 air changes per hour. Room temperature and humidity were monitored continuously and values outside of these ranges may have occasionally occurred, usually following room cleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC. The animals were provided with an automatically controlled light cycle of 12 hours light and 12 hours dark. Music was played during the light period.
Accommodation: Individually in stainless steel cages equipped with feed hoppers, drinking water bowls, with autoclaved wood (RCC Ltd, Fullinsdorf) and haysticks for gnawing.
Diet: Pelleted standard Provimi Kliba 3418 rabbit maintenance diet ad libitum (batch no. 33/00) provided by Provimi Kliba AG, CH-4303 Kaiseraugst. Results of analysis are archived at RCC Ltd, Itingen. Haysticks (QS no. 11/01) provided by Eberle Nafag AG, CH-9200 Gossau.
Water: Community tap water from Fullinsdorf, ad libitum. Results of bacteriological, chemical and contaminant analyses are archived at RCC Ltd, Itingen.

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Remarks:

test material moistened with approximately 0.1 ml bi-distilled water before application

Controls:

not required

Amount / concentration applied:

0.5 g (per animal) of test material was weighed as delivered by the sponsor and then moistened with bi-distilled water before application.

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

3

Details on study design:

Treatment:
Four days before treatment, the left flank was clipped with an electric clipper, exposing an area of approximately 100 cm² (10 cm x 10 cm). The skin of the animals was examined one day before treatment, and regrown fur of all animals was again clipped.
Animals with overt signs of skin injury or marked irritation which may have interfered with the interpretation of the results were not used in the test.
On the day of treatment, 0.5 g of test material was placed on a surgical gauze patch (ca. 2.5 cm x 2.5 cm). This gauze patch was applied to approximately 6 cm² of the intact skin of the clipped area. The patch was covered with a semi-occlusive dressing [Isoelast Heftpflaster Acryl 8 x 75 cm]. The dressing was wrapped around the abdomen and anchored with tape.
The duration of treatment was 4 hours. Then the dressing was removed and the skin was flushed with lukewarm tap water to clean the application site so that any reactions (erythema) were clearly visible at that time.

Observations:
Viability/Mortality: Daily from delivery of the animals to the termination of test.
Clinical signs: Daily from delivery of the animals to the termination of test.
Body weights: At start of acclimatization, on the day of application and at termination of observation.

Irritation Scores:
The skin reaction was assessed according to the numerical scoring system listed in the EEC Commission Directive 92/69/EEC, July 31, 1992 (based on the Draize score system) approximately 1, 24, 48 and 72 hours after the removal of the dressing, gauze patch and test item.
If evident, corrosive or staining properties of the test item were described and recorded.

Necropsy:
No necropsy was performed in the animals sacrificed at termination of observation.
All rabbits were sacrificed by an intravenous injection of NARCOREN (Rhône Mérieux GmbH, D-88471 Laupheim) into the ear vein at a dose of at least 1 ml/kg body weight (equivalent to 160 mg sodium pentobarbitone/kg body weight) and discarded.

No statistical analysis was performed.

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Irritant / corrosive response data:

Application of the test item to healthy intact rabbit skin resulted in a primary irritation index of 0.00.
The test item did not elicit any skin reactions at the application site of any animal at any of the observation times (all scores 0). The mean values from 24 to 72 hours were therefore 0 for erythema and 0 for oedema.
No staining by the test item of the treated skin was observed.
No irreversible alterations of the treated skin were observed nor were corrosive effects evident on the skin.

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.

Interpretation of results:

other: Not classified according to EU criteria

Conclusions:

According to Regulation (EC) No 1272/2008, the test material does not require classification as a skin irritant.

Executive summary:

In a skin irritation study (793642) according to EU Test Method B.4, the primary skin irritation potential of the test material was investigated by topical semi-occlusive application of 0.5g to 6 cm² intact left flank of each of three young adult New Zealand White rabbits. The duration of treatment was four hours. The scoring of skin reactions was performed 1, 24, 48 and 72 hours after removal of the dressing. The scores of each animal at the following reading times (24, 48, 72 hours) were used in calculating the respective mean values for each type of lesion.

The test item did not elicit any skin reactions at the application site of any animal at any of the observation times (all scores 0). The mean values from 24 to 72 hours were therefore 0 for erythema and 0 for oedema.

The test item caused no staining of the treated skin.

No corrosive effects were noted on the treated skin of any animal at any measuring interval.

According to Regulation (EC) No 1272/2008, the test material does not require classification as a skin irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 March 2001 - 26 March 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

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
- Source: Elevage Scientifique des Dombes. F-01400 Chatillon sur Chalaronne / France
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 2.4-2.5 kg
- Housing: Individually in stainless steel cages equipped with feed hoppers, drinking water bowls, with autoclaved wood (RCC Ltd, Fullinsdorf) and haysticks for gnawing.
- Diet: Pelleted standard Provimi Kliba 3418 rabbit maintenance diet ad libitum (batch no. 33/00) provided by Provimi Kliba AG, CH-4303 Kaiseraugst. Haysticks (QS no. 11/01) provided by Eberle Nafag AG, CH-9200 Gossau
- Water: Community tap water from Fullinsdorf, ad libitum.
- Acclimation: Under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Air-conditioned with target ranges for room temperature 20 ± 3 °C
- Humidity (%): relative humidity 30-70 %
- Air changes (per hr): approximately 10-14 air changes per hour
- Photoperiod (hrs dark / hrs light): The animals were provided with an automatically controlled light cycle of 12 hours light and 12 hours dark. Music was played during the light period.

Vehicle:

unchanged (no vehicle)

Controls:

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

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g

Duration of treatment / exposure:

On the day of treatment, the test material was placed in the conjunctival sac of the left eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of test item.

Observation period (in vivo):

1, 24, 48 and 72 hours, as well as 7 days after application.

Number of animals or in vitro replicates:

3 (1 male and 2 females)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The treated eyes were not rinsed after instillation.

SCORING SYSTEM: The ocular reaction was assessed according to the numerical scoring system listed in the EEC Commission Directive 92/69/EEC, July 31, 1992

TOOL USED TO ASSESS SCORE: Eye examinations were made with a Varta Cliptrix diagnostic-lamp (A. Riegger, Basel / Switzerland).

To evaluate the irritation potential of the test item (EEC Commission Directive 93/21/EEC, April 27, 1993), the mean values were calculated for each individual, using the scores between 24 and 72 hours.
The primary irritation score was calculated by totaling the mean cumulative scores at 24, 48 and 72 hours and then divided by the number of data points.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

mean

Time point:

24 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

mean

Time point:

48 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within:

Remarks:

7 days

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within:

Remarks:

7 days

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Application of the test item to healthy rabbit conjunctivae resulted in a primary irritation score of 0.56. The eye reactions (mean values from 24 to 72 hours) consisted of grade 0.00 corneal opacity, grade 0.00 iris lesions, grade 0.56 redness of the conjunctivae and grade 0.00 chemosis of the conjunctivae.

No abnormal findings were observed in the cornea or the iris in any animal at any time point. Slight reddening of the conjunctivae was noted in two animals from 1 to 24 hours after treatment. Slight to moderate reddening was also observed in the other animal from 1 to 72 hours after treatment. Slight swelling of the conjunctivae was evident in all animals at the 1-hour reading. Slight reddening of the sclera was observed in all animals at the 1-hour reading and in one female this persisted up to 24 hours after treatment. A slight watery discharge was present in one female at the 1-hour reading.

All eye reactions had cleared within 7 days after treatment. No staining of the treated eyes by the test item was observed. White remnants of the test item were evident in the eye or conjunctival sac of all animals 1 hour after treatment. No corrosion was observed at any of the measuring intervals.

Other effects:

No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.

BODY WEIGHTS

The body weights of all rabbits were considered to be within the normal range of variability.

Body weight in grams
Animal No.	Sex	First Day of Acclimatization	Day of Treatment	Last Day of Observation
88	male	2187	2468	2670
89	female	2063	2429	2648
90	female	2112	2423	2720

TABLE 1: EYE IRRITATION SCORES - INDIVIDUAL VALUES

Animal No.	Sex	Evaluation Interval	Corneal Opacity	Iris	Conjunctivae		Cumulative		Sclera
					Redness	Chemosis	Score	Mean
88	M	1 hour	0	0	1	1	2	2.33	1
89	F		0	0	1	1	2		1
90	F		0	0	2	1	3		1
88	M	24 hours	0	0	1	0	1	1	0
89	F		0	0	1	0	1		0
90	F		0	0	1	0	1		1
88	M	48 hours	0	0	0	0	0	0.33	0
89	F		0	0	0	0	0		0
90	F		0	0	1	0	1		0
88	M	72 hours	0	0	0	0	0	0.33	0
89	F		0	0	0	0	0		0
90	F		0	0	1	0	1		0
88	M	7 days	0	0	0	0	0	0	0
89	F		0	0	0	0	0		0
90	F		0	0	0	0	0		0

TABLE 2: EYE IRRITATION SCORES - MEAN VALUES AFTER 24, 48 AND 72 HOURS

Animal No.	Sex	Corneal Opacity	N	Iris	N	Conjunctivae				Primary Eye Irritation Score
						Redness	N	Chemosis	N
88	M	0	3	0	3	0.33	3	0	3	0.56
89	F	0	3	0	3	0.33	3	0	3
90	F	0	3	0	3	1	3	0	3
Mean score		0		0		0.56		0

N = number of available data points

TABLE 3: EYE IRRITATION SCORES - ASSESSMENT ACCORDING TO EEC GUIDELINES

Evaluated intervals	Corneal Opacity	Iris	Conjunctivae
			Redness	Chemosis
24 hours	Not Irritating	Not Irritating	Not Irritating	Not Irritating
48 hours
72 hours

Interpretation of results:

other: Not classified according to EU criteria

Conclusions:

According to Regulation (EC) No 1272/2008, the test material does not require classification as irritating to the eye.

Executive summary:

The primary eye irritation potential of the test material was investigated by instillation of 0.1 g into one eye of each of three young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours, as well as 7 days after test item application. The scores of each animal at the following reading times (24, 48 and 72 hours) were used in calculating the respective mean values for each type of lesion.

The primary irritation score was calculated by totaling the individual cumulative scores at 24, 48 and 72 hours and then dividing the resulting total by the number of data points. The primary irritation score was 0.56 (max. 13).

The eye reactions (mean values from 24 to 72 hours) consisted of grade 0.00 corneal opacity, grade 0.00 iris lesions, grade 0.56 redness of the conjunctivae and grade 0.00 chemosis of the conjunctivae.

No abnormal findings were observed in the cornea or the iris in any animal at any reading.

Slight reddening of the conjunctivae was noted in two animals from 1 to 24 hours after treatment. Slight to moderate reddening was also observed in the other animal from 1 to 72 hours after treatment. Slight swelling of the conjunctivae was evident in all animals at the 1-hour reading. Slight reddening of the sclera was observed in all animals at the 1-hour reading and in one female this persisted up to 24 hours after treatment.

A slight watery discharge was present in one female at the 1-hour reading.

White remnants of the test item were evident in the eye or conjunctival sac of all animals 1 hour after treatment.

No staining of the treated eyes by the test item was observed. No corrosion was observed at any of the measuring intervals. All eye reactions had cleared within 7 days after treatment.

According to Regulation (EC) No 1272/2008, the test material does not require classification as irritating to the eye.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 February 2011

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)

Deviations:

no

Principles of method if other than guideline:

The Enucleated Eye Test with isolated eyes of chickens has been recognized as a valuable alternative to the Draize eye irritation test, because it represents a test system nearest to the in vivo test, without the need to use live animals. In the Isolated Chicken Eye Test (ICET) the test compound is applied in one single dose onto the cornea of isolated eyes, which are obtained from slaughter animals after they have been killed. This method can provide detailed information about the effects of test items on the cornea, and is useful to compare products and to classify test items for regulatory use when they are severe irritants or corrosive to the eye. The test is described in OECD 438 and is approved by international regulatory agencies as a replacement for the identification of corrosives and severe irritants in the in vivo Rabbit eye assay (OECD 405).

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
Species of chicken: ROSS 308
Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út. 129.
Chicken heads were collected after slaughter in a commercial abattoir from chickens which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to LAB Research Ltd. at ambient temperature at the earliest convenience. After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at LAB Research Ltd. and processed within approximately 2 hours of collection.

Vehicle:

physiological saline

Controls:

other: one negative and three positve control eyes

Amount / concentration applied:

30mg Test mat. and imidazole(positive control)
30μL of isotonic saline (negative control)

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

The control eye and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ±5 minutes were considered acceptable.

The cornea thickness and cornea opacity were measured at all time points. Fluorescein retention was measured on two occasions, at base line (t=0) and 30 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

Seven eyes (three treated eyes, three positive control eyes, and one negative control eye)

Details on study design:

SELECTION AND PREPARATION OF EYES FOR THE TEST

Eyes selection:

After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 ml isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, 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.

Preparation of eyes:

The eye ball was carefully removed from the orbit by holding the nictitating membrane with a 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 so that the appropriate humidity was maintained.

Eyes examination and acclimatization time:

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 isotonic saline dripping from a stainless steel tube, at a rate of approximately 3 or 5 drops/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.

The appropriate number of eyes was selected and after being placed in the superfusion apparatus, were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for the eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and was conducted for approximately 45 to 60 minutes. A temperature of the circulating water was verified to ensured in all chambers were in the range of 32±1.5 °C during the acclimatization and treatment periods.

Identification:

The eyes were identified by chamber number, marked on the door of the chamber.

THE BASE LINE ASSESSMENTS
The base line assessments:

At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a base line (t=0) for each individual eye. The cornea thickness of the eyes should not increase by more than 5-7 % between the -45 and the zero time. Slight changes in thickness (0% to -3%) were observed in the eyes, this is considered normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Base line values were required to evaluate any potential test item related effects after treatment; the location of any minor findings were marked on the record sheet as a drawing. All eyes were considered to be suitable for the assay.


TEST PROCEDURE
Treatment:

After the zero reference measurements, take the first eye out of its chamber and place it on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position over a container to catch waste, while the test material was applied onto the centre of the cornea. The test material was applied in a volume of 30 mg by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance, taking care not to damage or touch the cornea with the application equipment. The positive control eyes were treated in a similar way with 30 mg Imidazole. The negative control eye was treated with 30μL of isotonic saline.

Test item removal:

The time of application was observed, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 ml isotonic saline at ambient temperature, taking care not to damage the cornea but attempting to remove all the residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum.

MEASUREMENTS AND THEIR METHODS

The cornea opacity measurement:

The slit lamp microscope should be focused so that the isotonic saline film should appear as a visible, clear (sharp) image on the cornea surface. The mechanical damage and cornea opacity can be seen in this position very well. The light intensity knob should be turned at 5 V position. The focal length can be changed by movement control knob on the mounting table. The following table give the other settings:

Slit length: Full length, Position 8
Slit width: Fully open, Position on indefinite

The cornea thickness measurement:

The first step, the slit lamp microscope should be focused that the isotonic saline should appear as a visible, clear (sharp) image on the cornea surface. Change the right ocular with the depth measuring ocular. The set light intensity knob to the 7.5 V position. Check that the light beam of the slit-lamp passes though the slit of the depth measuring device. The steel pin above the microscope eyepiece is where the device for cornea thickness measurements should be fitted. The following table specify the other settings:

Slit length: Full length, Position 8
Slit width: Fully open, Slit-width setting: 9.5

The fluorescein measurement:

One small drop of fluorescein solution 2 (w/v) % should be applied on the cornea surface for a few seconds, then rinsed off with isotonic saline. When fluorescein solution is used after the treatment, the eyes are not removed from the chambers, application and rinsing are performed in situ. The light intensity knob should be turned to the 5 V position. The settings are the same as for opacity assessment, but with the green light filter.

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

0.33

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

mean

Value:

0.67

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

mean up to 75 minutes

Value:

2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

mean up to 240 minutes

Value:

3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

The mean values of the treated eyes for maximum corneal thickness change, corneal opacity and fluorescein retention are given below. The conclusion on eye irritancy was based on the OECD guideline quantitative assessments.

Test Item: Test substance

Observation	Value	ICE Class*
Mean maximum corneal swelling at up to 75 min	2 %	I
Mean maximum corneal swelling at up to 240 min	3 %	I
Mean maximum corneal opacity	0.33	I
Mean fluorescein retention	0.67	II
Other Observations	The Test item was stuck on the cornea surface after the post-treatment rinse. The cornea surface was cleared 240 min after the post-treatment rinse.
Overall ICE Class*	2xI 1xII

 

In this in vitro eye irritation in the isolated chicken eyes test, the results suggest that the test item was not irritating. The test item was not a severe irritant.

Positive Control: Imidazole

Observation	Value	ICE Class*
Mean maximum corneal swelling at up to 75 min	8 %	II
Mean maximum corneal swelling at up to 240 min	18 %	III
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	2.83	IV
Other Observations	The Test item was stuck on the cornea surface after the post-treatment rinse. The cornea surface was not cleared 240 min after the post-treatment rinse.
Overall ICE Class*	1xIII 2xIV

The positive control Imidazole was classed as severely irritating, GHS Classification: Category 1. 

Negative Control: Sodium chloride

Observation	Value	ICE Class*
Mean maximum corneal swelling at up to 75 min	2 %	I
Mean maximum corneal swelling at up to 240 min	2 %	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.50	I
Other Observations	None
Overall ICE Class*	3xI

The negative control Sodium chloride 0.9% had no significant effects on the chicken eye in this study.

* Details of data interpretation for Isolated Chicken Eye Class are given in appendix 2 and 3.

TABLES OF ICE CLASSIFICATION

Table 2.1: ICE classification criteria for corneal thickness:

Mean Corneal Swelling (%)	ICE Class
0 to 5	I
>5 to 12	II
> 12 to 18 (>75 min after treatment)	II
> 12 to 18 (≥75 min after treatment)	III
>18 to 26	III
>26 to 32 (>75 min after treatment)	III
>26 to 32 (≥75 min after treatment)	IV
>32	IV

 

Table 2.2: ICE classification for opacity:

Mean Maximum Opacity Score	ICE Class
0.0 – 0.5	I
0.6 – 1.5	II
1.6 – 2.5	III
2.6 – 4.0	IV

 

Table 2.3: ICE classification criteria for mean fluorescein retention:

Mean Fluorescein Retention Score at 30 minutes post - treatment	ICE Class
0.0 – 0.5	I
0.6 – 1.5	II
1.6 – 2.5	III
2.6 – 4.0	IV

 

CLASSIFICATION

ASSESSMENT OF THE EX VIVO EYE IRRITANCY CLASSIFICATION

The following table is used to identify the probably eye irritancy potential of test items. In the case where the result indicates Severely Irritating, then the test item can be classified as Severe. In all other cases the probable level of irritancy can be reported, but a regulatory in vivo rabbit eye irritation test is required for regulatory classification and labelling purposes.

Classification (Combinations)	Combinations of the three ICE Classes^3
NI = not irritant	3×I 2×I, 1×II 2×II, 1×I
Slightly irritating	3×II 2×II, 1×III 1×I, 1×II, 1×III^1
Moderately irritating	3×III 2×III, 1×II 2xI, 1xIV^1 2×III, 1×IV^2 2×III, 1×I 2×II, 1×IV^1 1×II, 1×III, 1×IV^1
Severely irritating	3×IV 2×IV, 1×III 2×IV, 1×II^1 2×IV, 1×I^1 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)

¹: combinations of categories less likely to occur

²: combination can be considered a borderline case between irritating and severely irritating

³: The scheme provided is based on the INVITTOX protocol. Some modifications have been included in the scheme, provided by the author of the INVITTOX protocol; the scheme provided here is currently in the draft OECD guidance document on in vitro eye irritation.

Note: Small negative numbers for swelling following application are counted as zero (larger negative numbers due to erosion invalidate the swelling evaluation, but indicate a severe effect).

Interpretation of results:

other: Not classified according to EU criteria

Conclusions:

In this in vitro eye irritation study in the Isolated Chicken Eyes model, the results suggests that the test item was not irritating. According to the guideline OECD 438, the test substance does not require a classification as a severe eye irritant; an in vivo rabbit study is required for classification.

Executive summary:

An in vitro eye irritation study of the test item was performed in chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No.: 438 (7th September 2009).

After the zero reference measurements, the eye was held in horizontal position and 30 mg of the test substance was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with saline. The positive control eyes were treated in a similar way with 30 mg Imidazole. The negative control eye was treated with 30 μL of isotonic saline.

In this in vitro eye irritation study in the Isolated Chicken Eyes model with the test substance the results suggests that the test item was not irritating. According to the guideline OECD 438, the test substance does not require a classification as a severe eye irritant; an in vivo rabbit 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

Skin irritation

In a skin irritation study (793642) according to EU Test Method B.4, the primary skin irritation potential of the test material was investigated by topical semi-occlusive application of 0.5g to 6 cm² intact left flank of each of three young adult New Zealand White rabbits. The duration of treatment was four hours. The scoring of skin reactions was performed 1, 24, 48 and 72 hours after removal of the dressing. The scores of each animal at the following reading times (24, 48, 72 hours) were used in calculating the respective mean values for each type of lesion.

The test item did not elicit any skin reactions at the application site of any animal at any of the observation times (all scores 0). The mean values from 24 to 72 hours were therefore 0 for erythema and 0 for oedema.

The test item caused no staining of the treated skin.

No corrosive effects were noted on the treated skin of any animal at any measuring interval.

According to Regulation (EC) No 1272/2008, the test material does not require classification as a skin irritant.

Eye irritation

- In vitro study

An in vitro eye irritation study of the test item was performed in chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No.: 438 (7th September 2009).

After the zero reference measurements, the eye was held in horizontal position and 30 mg of the test substance was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with saline. The positive control eyes were treated in a similar way with 30 mg Imidazole. The negative control eye was treated with 30 μL of isotonic saline.

In this in vitro eye irritation study in the Isolated Chicken Eyes model with the test substance the results suggests that the test item was not irritating. According to the guideline OECD 438, the test substance does not require a classification as a severe eye irritant; an in vivo rabbit study is required for classification.

- In vivo study

The primary eye irritation potential of the test material was investigated by instillation of 0.1 g into one eye of each of three young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours, as well as 7 days after test item application. The scores of each animal at the following reading times (24, 48 and 72 hours) were used in calculating the respective mean values for each type of lesion.

The primary irritation score was calculated by totaling the individual cumulative scores at 24, 48 and 72 hours and then dividing the resulting total by the number of data points. The primary irritation score was 0.56 (max. 13).

The eye reactions (mean values from 24 to 72 hours) consisted of grade 0.00 corneal opacity, grade 0.00 iris lesions, grade 0.56 redness of the conjunctivae and grade 0.00 chemosis of the conjunctivae.

No abnormal findings were observed in the cornea or the iris in any animal at any reading.

Slight reddening of the conjunctivae was noted in two animals from 1 to 24 hours after treatment. Slight to moderate reddening was also observed in the other animal from 1 to 72 hours after treatment. Slight swelling of the conjunctivae was evident in all animals at the 1-hour reading. Slight reddening of the sclera was observed in all animals at the 1-hour reading and in one female this persisted up to 24 hours after treatment.

A slight watery discharge was present in one female at the 1-hour reading.

White remnants of the test item were evident in the eye or conjunctival sac of all animals 1 hour after treatment.

No staining of the treated eyes by the test item was observed. No corrosion was observed at any of the measuring intervals. All eye reactions had cleared within 7 days after treatment.

According to Regulation (EC) No 1272/2008, the test material does not require classification as irritating to the eye.

Justification for classification or non-classification

By reference to the data summarised within this dataset, the substance does not meet the criteria for classification for irritation according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.