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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-27 to 2018-03-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
A temperature higher than 23°C was registered on 08 January 2018. The maximum value measured was 25°C. This deviation is considered as without impact on the conclusion of the study.
Principles of method if other than guideline:
At the request of the Ethics Committee, the following procedure, slightly different from the
recommendation of the OCDE guideline, has been followed:
• Sixty minutes prior to test substance application (TSA), buprenorphine (batch No.: H011) 0.01
mg/kg was administered by subcutaneous injection (SC) to provide a therapeutic level of systemic
analgesia.
• Five minutes prior to TIA, one or two drops of a topical ocular anesthetic (e.g. Tetracaïne 1% batch
No.: 7D79A) were applied to each eye. The eye of each animal that was not treated with a test article,
but which was treated with topical anesthetics, served as a control.
• Eight hours after TSA, buprenorphine 0.01 mg/kg SC and meloxicam (batch No.: 16A271)
0.5 mg/kg SC were administered to provide a continued therapeutic level of systemic analgesia.
• About 24 hours after treatment, after the observations of the animal, if the animal presents with
marked lesions or if suffering of the animal was noted, buprenorphine 0.01 mg/kg SC and meloxicam
0.5 mg/kg SC were administered (Cf Analgesia administration appendix 2).
Remark: the lesions are considered as marked if the score obtained is ≥ 2 for at least two parameters
chemosis, redness or opacity of the cornea.
• Up to the end of the study and after each observation of the animal, buprenorphine 0.01 mg/kg SC
and meloxicam 0.5 mg/kg SC were administered if suffering or marked lesions were observed.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals / tissue source

Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Animals
One female albino New Zealand rabbit was supplied by Hypharm (F-49450 La Renaudiere). The
animal was kept during a minimal 5-day acclimatisation period.
At the beginning of the test, the animal was 15 weeks old.
It was identified prior to inclusion in the test by means of a numbered ring on the edge of one ear.

Housing
The animal was kept in an individual box installed in conventional air conditioned animal husbanding:
The temperature and relative humidity of the main test were controlled to remain within target ranges
of 17°C to 23°C and 30% to 70%, respectively.
The rate of air exchange was at least ten changes per hour and the lighting was controlled by a time
switch to give twelve hours continuous light (07.00 to 19.00) and twelve hours darkness.

Food and drink
Drinking water (tap-water from public distribution system) and foodstuff (ENVIGO – 2930C) were
supplied ad libitum.
Microbiological and chemical analyses of the water were carried out once every six months by Bureau
Veritas – Eurofins (FRANCE).

Test system

Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
0.1 mL of the test item was instilled, as supplied
Duration of treatment / exposure:
n.a.
Observation period (in vivo):
Ocular examinations were performed on both right and left eyes 1 hour, 24, 48 and 72 hours following
treatmentIf no reaction is observed 72 hours after instillation, the study is terminated. In case of persistent reactions,
additional observations can be carried out from D7 to D21 in order to determine the reversible character of the
lesions observed.
Number of animals or in vitro replicates:
One female
Details on study design:
Treatment
0.1 mL of the test item was instilled, as supplied, into the conjunctival sac of one eye after gently
pulling the lower lid away from the eyeball. The lids were then gently held together for about one
second in order to prevent loss of the test item. The other eye remained untreated serving as control.
Initially, a single animal was treated. After consideration of the responses in the first animal, no
additional animal was treated.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3.3
Max. score:
4
Reversibility:
not fully reversible within: 21 d
Remarks on result:
positive indication of irritation
Remarks:
for detailed information please see "Any other information on results incl. tables"
Irritation parameter:
overall irritation score
Basis:
animal #1
Time point:
21 d
Score:
24
Max. score:
90
Reversibility:
not fully reversible within: 21d
Remarks on result:
positive indication of irritation
Remarks:
for detailed information please see "Any other information on results incl. tables"
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
21 d
Score:
4
Max. score:
20
Reversibility:
not fully reversible within: 21d
Remarks on result:
positive indication of irritation
Remarks:
for detailed information please see "Any other information on results incl. tables"
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 21d
Remarks on result:
positive indication of irritation
Remarks:
for detailed information please see "Any other information on results incl. tables"
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
not fully reversible within: 21d
Remarks on result:
positive indication of irritation
Remarks:
for detailed information please see "Any other information on results incl. tables"
Irritant / corrosive response data:
The ocular reactions observed during the study have been moderate to severe and partially reversible:
- at the conjunctivae level: an important redness was noted 1 hour after the test item instillation and
remaining on the last day of the test (day 21) with slight intensity (grade 1). This reaction was
associated with a severe chemosis noted 24 hours after the test item instillation and remaining on the
last day of the test (day 21) with slight intensity (grade 1).
- at the iris level: an injection noted 1 hour after the test item instillation and haemorrhage was noted
between days 1 and 7 (grade 2).
- at the corneal level: an important opacity, noted 24 hours after the test item instillation and remaining
on the last day of the test (day 21) with moderate intensity (grade 2).
Withe secretions requiring a physiological saline rinse were noted between days 1 and 7.
A corneal-neovascularisation was noted between days 14 and 21.
Brown coloration on eyelids and nictitating membrane was noted between days 0 and 7.

Any other information on results incl. tables

EVALUATION OF OCULAR IRRITATION

Eye examinations are carried out using the scale of lesion scores in the following order:

 

CHEMOSIS (A)

·       No swelling.............................................................................................................  0

·       Slight swelling, including the nictitating membrane.................................................  1

·       Swelling with eversion of the eyelid.........................................................................  2

·       Swelling with eyelid half-closed...............................................................................  3

·       Swelling with eyelid more than half-closed..............................................................  4

 

 

DISCHARGE (B)

·     No discharge...........................................................................................................  0

·     Slight discharge (normal slight secretions in the inner corner not to be taken into account).................................. 1

·     Discharge with moistening of the eyelids and neighbouring hairs..............................  2

·     Discharge with moistening of the eyelids and large areas around the eye...................  3

 

 

REDNESS (C)

·     Blood vessels normal...............................................................................................  0

·     Vessels significantly more prominent than normal...................................................  1

·     Vessels individually distinguishable with difficulty

-...Generalised red coloration...................................................................................  2

-...Generalised deep red coloration...........................................................................  3

 

 

Iris (D)

·     Normal...................................................................................................................  0

·     Iris significantly more wrinkled than normal, congestion, swelling of the iris which continues to react to light, even slowly.............................  1

·     No reaction to light, haemorrhage, significant damage (any or all of these characteristics)...........................................................................  2

 

 

Cornea:DEGREE OF OPACITY (E)

·     No modification visible either directly or after instillation of fluorescein (no loss of glint or polish)........................  0

·     Translucent areas (diffuse or disseminated), iris details clearly visible........................  1

·     Easily identifiable translucent area, iris details slightly obscured...............................  2

·     Opalescent area, no iris details visible, pupil outline scarcely distinguishable.............  3

·     Total corneal opacity, completely obscuring the iris and pupil...................................  4

 

 

Cornea:EXTENT OF OPACITY (F)

·     Opaque area present but covering one quarter or less................................................  1

·     Between one quarter and half..................................................................................  2

·     Between half and three quarters...............................................................................  3

·     Between three quarters and the entire surface...........................................................  4

Table 1

 Animal No     Time after treatment     Conjunctivae    Iris  Cornea
 Chemosis (A) Redness (C)  Lesion (D)  Opacity (E) 
 #1 (A6838)              24 h  4  3  2  3
 48 h  3  3  2  3
 72 h  3  3  2  3
 TOTAL  10  9  6  9
 MEAN  3.3  3  2  3

Table 2

 Observation time    CONJUNCTIVAE          IRIS     CORNEA        Individual irritation index    
 A (A+B+C)x2  Dx5  ExFx5 
         X=   Y=      Z=  X+Y+Z= 
 1 hour (D0)  2  3  3 16  20  41 
 24 hours (D1)  3  20 10  60  90 
 48 hours (D2)  3  1  14 10  60  84 
 72 hours (D3)  1  14 10  60  84 
 Day 7 (D7)  2  0  2  12 10  3 45  67 
 Day 14 (D14)  2  0  2  8 20  28 
 Day 21 (D21)  1  0  1  4 20  24 

FromT0 to T0+30 sec: increase of salivation associated with tremors

T0+1h: decrease of spontaneous activity

D1 to D7:haemorrhage

D1 to D7: brown coloration on lids and nictitating membrane

D1 to D7:withe secretions requiring a physiological saline rinse,cornealoedema.

D2 to D7:scab on internal and external eyelids.

D14 to D21:corneal-neovascularisation

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The results obtained, under these experimental conditions, enable to conclude that the test item (S)-Nicotine has to be classified in category 1 “irreversible effects on the eye” in accordance with the Regulation (EC) No. 1272/2008, the test item.
The signal word “Danger” and hazard statement H318 “Causes serious eye damage” are required.
Executive summary:

The test item (S)-Nicotine was instilled, as supplied, into the eye of one New Zealand rabbit at the dose of 0.1 mL. The experimental protocol was established on the basis of the official method as defined in the O.E.C.D. Test Guideline No. 405 dated October 9th, 2017.

An increase of salivation associated with tremors was noted immediately after the test item instillation and this during approximately 30 seconds. A decrease of spontaneous activity was noted at 1 hour after the test item instillation. The animal recovered a normal activity on day 1.

The ocular reactions observed during the study have been moderate to severe and partially reversible:

- at the conjunctivae level: an important redness was noted 1 hour after the test item instillation and

remaining on the last day of the test (day 21) with slight intensity (grade 1). This reaction was

associated with a severe chemosis noted 24 hours after the test item instillation and remaining on the

last day of the test (day 21) with slight intensity (grade 1).

- at the iris level: an injection noted 1 hour after the test item instillation and haemorrhage was noted

between days 1 and 7 (grade 2).

- at the corneal level: an important opacity, noted 24 hours after the test item instillation and remaining

on the last day of the test (day 21) with moderate intensity (grade 2).

Withe secretions requiring a physiological saline rinse were noted between days 1 and 7.

A corneal-neovascularisation was noted between days 14 and 21.

Brown coloration on eyelids and nictitating membrane was noted between days 0 and 7.

In conclusion, the results obtained, under these experimental conditions, enable to conclude that the

test item (S)-Nicotine has to be classified in category 1 “irreversible effects on the eye” in

accordance with the Regulation (EC) No. 1272/2008, the test item.

The signal word “Danger” and hazard statement H318 “Causes serious eye damage” are required.