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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:
19 June 2000 to 27 June 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The standard information requirements for REACH Annex VII substances for serious eye damage/eye irritation can be satisfied by an assessment of the available in vivo data, physicochemical properties (i.e. acid or alkali reserve) or in vitro eye irritation studies. Conducted prior to the validation and regulatory acceptance of alternative methods, a reliable (Klimisch 1) and GLP compliant in vivo Draize test was available for the test substance and considered sufficient to fulfil the information requirement.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
1987
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Caryophyllene
EC Number:
201-746-1
EC Name:
Caryophyllene
Cas Number:
87-44-5
Molecular formula:
C15H24
IUPAC Name:
(1R,4E,9S)-4,11,11-trimethyl-8-methylidenebicyclo[7.2.0]undec-4-ene

Test animals / tissue source

Species:
rabbit
Strain:
other: Chbb:HM (SPF)
Remarks:
albino
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 2.7 to 2.9 kg b.w.
- Housing: Caged individually in PPO cages (floor area: 2576 sq.cm) with perforated floor
- Diet: Pelleted complete rabbit diet, ad libitum (Altromin 2123)
- Water: Domestic quality drinking water acidified with hydrochloric acid to pH 2.5, ad libitum
- Acclimation period: At least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.1 mL
Duration of treatment / exposure:
Single exposure
Observation period (in vivo):
At 1, 24, 48 and 72 hours and 7 days after treatment
Number of animals or in vitro replicates:
4
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: Eyes were rinsed with 20 mL 0.9% sodium chloride solution
- Time after start of exposure: Eyes were rinsed 24 hours after exposure

SCORING SYSTEM:
When Fluorescein was used the cornea reactions were scored both before and alter the instillation of Fluorescein.

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

Area of cornea involved
1: One quarter (or less) but not zero.
2: More than 1 quarter, but less than half.
3: More than half, but less than 3 quarters.
4: More than 3 quarters, up to whole area.

Iris
0: Normal.
1: Markedly deepened folds, congestion, swelling, moderate circumcorneal injection (any of these or combination of any thereof), iris still reacts to light (sluggish reaction is positive).
2: No reaction to light, haemorrhage, gross destruction (any or all of these).

Conjunctiva redness (refers to palpebral and bulbar conjunctivae excluding cornea and Iris)
0: Vessels normal.
1: Some vessels definitely injected.
2: Diffuse, crimson red, individual vessels not easily discernible.
3: Diffuse beefy red.

Conjunctiva chemosis:
0: No swelling.
1: Any swelling above normal (includes nictitating membrane).
2: Obvious swelling with partial eversion of lids.
3: Swelling with lids about half closed.
4: Swelling with lids more than half closed.

Conjunctiva discharge:
0: No discharge.
1: Any amount different from normal (does not include small amounts observed in inner canthus of normal animals).
2: Discharge with moistening of the lids and hairs just adjacent to lids.
3: Discharge with moistening of the lids and hairs, and considerable area around the eye.

TOOL USED TO ASSESS SCORE: fluorescein

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Basis:
animal #1
Remarks:
(1813)
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Remarks:
(1815)
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #3
Remarks:
(1816)
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #4
Remarks:
(1817)
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
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:
animal #1
Remarks:
(1813)
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Remarks:
(1815)
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #3
Remarks:
(1816)
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #4
Time point:
24/48/72 h
Score:
0
Max. score:
1
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
1
Reversibility:
fully reversible
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Remarks:
(1813)
Time point:
24/48/72 h
Score:
1.33
Max. score:
2
Reversibility:
fully reversible within: 7 d
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Remarks:
(1815)
Time point:
24/48/72 h
Score:
1.33
Max. score:
2
Reversibility:
fully reversible within: 7 d
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #3
Remarks:
(1816)
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 72 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #4
Remarks:
(1817)
Time point:
24/48/72 h
Score:
1.33
Max. score:
2
Reversibility:
fully reversible within: 7 d
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
mean
Time point:
24/48/72 h
Score:
1.2
Max. score:
2
Reversibility:
fully reversible within: 7 d
Remarks on result:
probability of weak irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
(1813)
Time point:
24/48/72 h
Score:
0.67
Max. score:
2
Reversibility:
fully reversible within: 72 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
(1815)
Time point:
24/48/72 h
Score:
0.67
Max. score:
2
Reversibility:
fully reversible within: 72 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Remarks:
(1816)
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 7 d
Remarks on result:
probability of weak irritation
Irritation parameter:
chemosis score
Basis:
animal #4
Remarks:
(1817)
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 72 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0.8
Max. score:
2
Reversibility:
fully reversible within: 7 d
Remarks on result:
probability of weak irritation
Irritant / corrosive response data:
One hour following application of the test article all four animals exhibited a diffuse, crimson red conjunctiva with individual vessels not easily discernible with partial eversion of lids. Animal 1 (#1813) and Animal 4 (#1817) also presented a discharge and an iris with markedly deepened folds, congestion, swelling, moderate circumcorneal injection, respectively. The mean values for corneal opacity, iris lesions, redness of conjunctivae and oedema of conjunctivae (chemosis) were 0.0, 0.0, 1.2 and 0.8, respectively. All weak signs of irritation were reversible and 7 days after application of the test article all four animals were free of any signs of eye irritation.

Any other information on results incl. tables

Table 1. Scores for ocular lesions

 Ocular lesion  Animal number  1 h  24 h (after Fluorescein)  48 h

 72 h

  7 days

 Cornea opacity, degree

 1

 0

 0 (0)

 0

 0

  0
 0  2  0  0 (0)  0  0  0
   3  0  0 (0)  0  0  0
   4  0  0 (0)  0  0  0
 Cornea opacity, area  1  0  0 (0)  0  0  0
   2  0  0 (0)  0  0  0
   3  0  0 (0)  0  0  0
   4  0  0 (0)  0  0  0
 Iris  1  0  0  0  0  0
   2  0  0  0  0  0
   3  0  0  0  0 0 
   4  1  0  0  0  0
 Conjunctiva redness  1  2  2  1  1  0
   2  2  2  1  1  0
   3  2  2  1  0  0
   4  2  2  1  1  0
 Conjunctiva chemosis  1  2  1  1  0  0
   2  2  1  1  0  0
   3  2  1  1  1  0
   4  2  2  1  0  0
 Conjunctiva discharge  1  1  0  0  0  0
   2  0  0  0  0  0
   3  0  0  0  0  0
   4  0  0  0  0  0

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The mean values for corneal opacity, iris lesions, redness of conjunctivae and oedema of conjunctivae (chemosis) were 0.0, 0.0, 1.2 and 0.8, respectively. All weak signs of irritation were reversible and 7 days after application of the test article all four animals were free of any signs of eye irritation. The test item should not be classified as irritating to the eye, according to the directive of the EEC commission 93/21/EEC and GHS/EU CLP criteria.
Executive summary:

The study was initiated with one rabbit, since no marked eye irritation was observed, another three rabbits were included. An aliquot of 0.1 mL test item was instilled into the conjunctival sac of one eye of each rabbit, while the other eye remained untreated and was used as the negative control. Assessments of damage/irritation (cornea opacity, iris lesion, conjunctival redness and conjunctival oedema) were made 1, 24, 48 and 72 hours, in addition to 7 days, following treatment.

Weak conjunctival inflammation, accompanied by slight swelling was observed in all four rabbits for up to 72 hours after instillation. The mean values for corneal opacity, iris lesions, redness of conjunctivae and oedema of conjunctivae (chemosis) were 0.0, 0.0, 1.2 and 0.8, respectively. All weak signs of irritation were reversible and 7 days after application of the test article all four animals were free of any signs of eye irritation. The test material should not be classified as irritating to the eye, according to the directive of the EEC commission 93/21/EEC (4 May 1993) and GHS/EU CLP criteria. Conducted according to OECD TG 405 (EU Method B.5) and GLP, the study was considered to be reliable (Klimisch 1).