Reaction mass of 3-phenyl-6-[(3-phenyl-3,4-dihydro-2H-1,3-benzoxazin-8-yl)methyl]-3,4-dihydro-2H-1,3-benzoxazine and 6,6'-methanediylbis(3-phenyl-3,4-dihydro-2H-1,3-benzoxazine)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date: 07 March 2008 and Experimental completion date: 17 March 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

6,6'-(1-methylethylidene)bis[3,4-dihydro-3-phenyl-2H-1,3-benzoxazine

EC Number:

604-960-7

Cas Number:

154505-70-1

Molecular formula:

C31 H30 N2 O2

IUPAC Name:

6,6'-(1-methylethylidene)bis[3,4-dihydro-3-phenyl-2H-1,3-benzoxazine

Test material form:

solid

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

- Animals: Young Adult New Zealand White Rabbit, SPF
- Age when Treated: 18 weeks (male) and 14 weeks (females)
- 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. 75 and Female Nos. 76, 77.

Conditions:
Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environment with ranges for room temperature 17-23 °C and for relative humidity between 30-70 % (values above 70 % during cleaning process possible), automatically controlled light cycle of 12 hours light and 12 hours dark, music during the daytime light period.

Accommodation:
Individually in stainless steel cages equipped with feed hoppers and drinking water bowls. Wood blocks (RCC Ltd, Füllinsdorf) and haysticks 4642 (batch no. 36/07, Provimi Kliba AG) were provided for gnawing.

Diet:
Pelleted standard Provimi Kliba 3418 rabbit maintenance diet ad libitum (batch no. 70/07) provided by Provimi Kliba AG, CH-4303 Kaiseraugst.

Water:
Community tap water from Füllinsdorf ad libitum.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

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

Amount / concentration applied:

0.1 gram of the test item.

Duration of treatment / exposure:

One single application

Observation period (in vivo):

72 hours after administration.

Number of animals or in vitro replicates:

3 ( Animals of both sexes were used)

Details on study design:

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

TOOL USED TO ASSESS SCORE: Eye examinations were made with a Varta Cliptrix disgnostic-lamp (Roth AG, CH-4153 Reinach/Switzerland).

SCORING SYSTEM:
COMMISSION DIRECTIVE 2004/73/EC, APRIL 29, 2004
Grading of Ocular Lesions

Cornea
Opacity: degree of density (area most dense taken for reading)
- No ulceration or opacity 0
- Scattered or diffuse areas of opacity (other than slight dulling of normal luster), details of iris clearly visible 1
- Easily discernible translucent area, details of iris slightly obscured 2
- Nacreous area, no details of iris visible, size of pupil barely discernible 3
- Opaque cornea, iris not discernible through the opacity 4

Area of cornea involved
- Zero 0
- One quarter (or less) but not zero 1
- Greater than one quarter, but less than half 2
- Greater than half, but less than three quarters 3
- Greater than three quarters, up to whole area 4

Iris
- Normal 0
- Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperemia, or injection, any of these or combination of any thereof, iris
still reacting to light (sluggish reaction is positive) 1
- No reaction to light, hemorrhage, gross destruction (any or all of these) 2

Conjunctivae
Redness (refers to most severe reading of palpebral and bulbar conjunctivae when compared with control eye)
- Blood vessels normal 0
- Some blood vessels definitely hyperemic (injected) 1
- Diffuse, crimson color, individual vessels not easily discernible 2
- Diffuse beefy red 3

- Chemosis: lids and/or nictitating membranes No swelling 0
- Any swelling above normal (including nictitating membranes) 1
- Obvious swelling with partial eversion of lids 2
- Swelling with lids about half-closed 3
- Swelling with lids more than half-closed 4

ADDITIONALLY, OCULAR DISCHARGE, REDDENING OF THE SCLERAE AND STAINING OF CONJUNCTIVAE, SCLERAE AND CORNEA BY THE TEST ITEM WAS ASSESSED ACCORDING TO THE FOLLOWING SCHEME:

Ocular Discharge
- No discharge 0
- Slight: Any amount different to normal (does not include small amount observed in inner canthus of normal animal) 1
- Moderate: Discharge with moistening of the lids and hair just adjacent to the lids 2
- Marked: Discharge with moistening of the lids and hairs, and a considerable area around the eye (running) 3

Sclerae
Redness
- Normal: Blood vessels normal 0
- Slight reddening: Some blood vessels definitely hyperemic (injected) 1
- Moderate reddening: Diffuse, crimson color, individual vessels not easily discernible 2
- Marked reddening: Diffuse beefy red 3

Staining of Conjunctivae, Sclerae and Cornea by the Test Item
- Not observed 0
- Slight staining 1
- Marked staining 2

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

Animal Number 75/76/77 (M/F/F)

Time point:

24/48/72 h

Score:

0

Max. score:

0

Results 2

Irritation parameter:

iris score

Basis:

mean

Remarks:

Animal Number 75/76/77 (M/F/F)

Time point:

24/48/72 h

Score:

0

Max. score:

0

Results 3

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

Animal Number 75/76/77 (M/F/F)

Time point:

24/48/72 h

Score:

0

Max. score:

0

Results 4

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

Animal number 75 (Male)

Time point:

24/48/72 h

Score:

0

Max. score:

0

Results 5

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

Animal number 76 (Female)

Time point:

24/48/72 h

Score:

0

Max. score:

0

Results 6

Irritation parameter:

conjunctivae score

Basis:

animal #3

Remarks:

Animal number 77 (Female)

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

Irritation
The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity and iris were 0.00 for all three animals. The individual mean scores for the conjunctivae were 0.00, 0.00 and 0.33 for reddening and 0.00, for chemosis for all three animals.
No abnormal findings were observed in the cornea or iris of any animal at any of the measurement intervals.
A moderate reddening of the conjunctivae was noted in all animals at the 1-hour observation and persisted as slight reddening in one female up to the 24-hour reading. A slight swelling (chemosis) of the conjunctivae was observed in all animals 1 hour after treatment. Additionally, a slight to moderate reddening of the sclerae was present 1 hour after instillation in all animals and persisted in the male animal up to the 24-hour reading. Furthermore, a slight discharge was recorded in all animals at the 1-hour reading.
No abnormal findings were observed in the treated eye of any animal 48-hours after treatment.

Corrosion
No corrosion of the cornea was observed at any of the reading times.

Other effects:

- Viability / Mortality / Clinical Signs: No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.
- Coloration: No staining produced by the test item of the treated eye was observed.
- Test Item Remnants: Yellow test item remnants were evident in the eye or conjunctival sac of each animal 1 hour after treatment.
- Body weights: The body weights of all rabbits were considered to be within the normal range of variability.

Any other information on results incl. tables

Body weights in grams.

Animal N°	Sex	First day of acclimatization	Day of treatment	Last day of observation
75	male	3037	3092	3168
76	female	2849	2974	3171
77	female	2805	2866	3044

Eye Irritation Scores – Individual Values

Animal number	Sex	Evaluation Interval*	Corneal Opacity	Area of corneal opacity	Iris	Redness	Chemosis	Sclera
75	M	1 hour	0	0	0	2	1	2
76	F	1 hour	0	0	0	2	1	1
77	F	1 hour	0	0	0	2	1	2
75	M	24 hours	0	0	0	0	0	1
76	F	24 hours	0	0	0	0	0	0
77	F	24 hours	0	0	0	1	0	0
75	M	48 hours	0	0	0	0	0	0
76	F	48 hours	0	0	0	0	0	0
77	F	48 hours	0	0	0	0	0	0
75	M	72 hours	0	0	0	0	0	0
76	F	72 hours	0	0	0	0	0	0
77	F	72 hours	0	0	0	0	0	0

* Examinations were performed at the specified times after instillation of the test item.

Eye Irritation Scores – Individual Mean Values after 24, 48 and 72 Hours

Animal number	Sex	Corneal Opacity	N	Iris	N	Redness	N	Chemosis	N
75	M	0.00	3	0.00	3	0.00	3	0.00	3
76	F	0.00	3	0.00	3	0.00	3	0.00	3
77	F	0.00	3	0.00	3	0.33	3	0.00	3

N= number of avilable data points

Eye Irritation Scores – Assessment According to EC Guidelines

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

Individual Findings (For animal N° 75, 76 and 77.

After 1 hour

Cornea: NO ABNORMAL FINDINGS NOTED

Iris: NO ABNORMAL FINDINGS NOTED

Conjunctivae: Moderately reddened / Slight swelling

Discharge: Slight discharge

Sclerae: Moderatley reddened

Test item: Yellow remnants evident

After 24 hours:

Cornea: NO ABNORMAL FINDINGS NOTED

Iris: NO ABNORMAL FINDINGS NOTED

Conjunctivae: NO ABNORMAL FINDINGS NOTED

Discharge: NO ABNORMAL FINDINGS NOTED

Sclerae: Slightly reddened

Test item: NO REMNANTS EVIDENT

After 48 hours:

Cornea: NO ABNORMAL FINDINGS NOTED

Iris: NO ABNORMAL FINDINGS NOTED

Conjunctivae: NO ABNORMAL FINDINGS NOTED

Discharge: NO ABNORMAL FINDINGS NOTED

Sclerae: NO ABNORMAL FINDINGS NOTED

Test item: NO REMNANTS EVIDENT

After 72 hours:

Cornea: NO ABNORMAL FINDINGS NOTED

Iris: NO ABNORMAL FINDINGS NOTED

Conjunctivae: NO ABNORMAL FINDINGS NOTED

Discharge: NO ABNORMAL FINDINGS NOTED

Sclerae: NO ABNORMAL FINDINGS NOTED

Test item: NO REMNANTS EVIDENT

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based upon the referred classification criteria (Commission Directive 2001/59/EC of August 06, 2001), the test item is considered to be “not irritating” to the rabbit eye.

Executive summary:

The primary eye irritation potential of the test item was investigated according to OECD test guideline no. 405. The test item was applied by instillation of 0.1 g into the left eye of each of three young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours after test item instillation.

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity and iris were 0.00 for all three animals. The individual mean scores for the conjunctivae were 0.00, 0.00 and 0.33 for reddening and 0.00 for chemosis for all three animals.

The instillation of the test item into the eye resulted in early-onset and transient ocular changes, such as reddening of the conjunctivae and sclerae, discharge and chemosis. These effects were reversible and were no longer evident 24 or 48 hours after treatment. No abnormal findings were observed in the cornea or iris of any animal at any of the examinations. No corrosion was observed at any of the measuring intervals. Yellow test item remnants were evident in the eye or conjunctival sac of each animal 1 hour after treatment. No staining of the treated eyes by the test item was observed and no clinical signs were observed.

Thus, the test item did not induce significant or irreversible damage to the rabbit eye.

Based upon the referred classification criteria (Commission Directive 2001/59/EC of August 06, 2001), the test item is considered to be “not irritating” to the rabbit eye.