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Description of key information

One in vivo skin irritation study (using method similar or equivalent to OECD Guideline 404) was performed using bis(2-3-epoxypropyl)cyclohex-4-ene-1-2-dicarboxylate in Himalayan rabbits. 0.5 mL of the undiluted test substance was applied to the shaved/and or not scarified skin under occlusive dressing during 24 hours. The primary skin irritation index in this experiment was established to be 3.0. The test material caused therefore a moderate skin irritation to rabbits.

Under the conditions of this experiment the test material was found to be classified as Category 2 (irrittating to skin) based on GHS criteria.

One in vivo eye irritation study was performed (using method similar or equivalent to OECD Guideline 405 using bis(2-3-epoxypropyl)cyclohex-4-ene-1-2-dicarboxylate in Himalayan rabbits.

0.1 mL of the test substance was placed into the conjunctival sac of the left eye of each animal. The right eye was not treated and served as an untreated control. In 3 of the 6 rabbits approximately 30 seconds after treatemnt the treated eyes was flushed with 10 mL of lukewarm water.

In the present experiment the iritaion index was established as 9.2 for cornea, 0 for Iris and 5.6 for the conjunctivae in not rinsed eyes after treatment.

Rinsing the eyes following instillation was of very good effect.

Under the conditions of this experiment the test material was found to be classified as Category 2 (irritating to eyes) based on GHS criteria.

Rinsing the eyes following instillation was of very good effect.

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:
1977
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
other: The procedure used was the patch test technique described in the "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959), the US Association of Food and Drug Officials (AFDO).
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
not applicable
GLP compliance:
no
Species:
rabbit
Strain:
Himalayan
Details on test animals or test system and environmental conditions:
The test was performed on 3 male and 3 female rabbits of the Himalayan breed weighing 1.5 to 2 kg.
They were housed individually in metal cages, were kept at a constant room temperature of 22 + 1 C, at a relative humidity of 55 + 5 % and on a 10 hours light cycle day.
The animals received ad libitum Standard rabbit food - NAFAG, Gossau SG - and water. Prior to treatment they were adapted to our laboratories for a minimum of 4 days.
Type of coverage:
occlusive
Preparation of test site:
other: 2 days before treatment: shaved and immediately before treatment the left side was slightly scarified.
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
0.5 mL.
Duration of treatment / exposure:
24 hours exposure time.
Observation period:
72 hours
Number of animals:
3 male and 3 female
Details on study design:
Two days before treatment the entire back and the flank of the rabbits were shaved with an electric clipper and immediately before treatment the shaven skin on the left side was slightly scarified. For treatment gauze patches of 2.5 x 2.5 cm with test material were applied to the prepared abraded and non abraded skin, in quantities of 0.5 ml.

The patches were covered with an impermeable material and were fastened to the body of the rabbit with adhesive tape. The dressings were removed after a 24 hour exposure.
The skin irritation was scored immediately and 48 hours after removal of the dressings (24 and 72 hours after
initiation of treatment). Skin irritation was scored in accordance with the irritation score according to the scoring system available below.

The severity of skin irritation was rated as follows:
A mean value of less than 2 indicated mild, from 2 to 6 moderate and above 6 severe skin irritation.

SCORING SYSTEM:

Score for skin irritation in rabbits
Erythema and eschar formation
- No erythema: 0
- Very slight erythema (barely perceptible): 1
- Well defined erythema: 2
- Moderate to severe erythema: 3
- Severe erythema (beet redness) to slight eschar formation (injuries in depth): 4

Maximum possible erythema score: 4

Edema formation
- No edema: 0
- Very slight edema (barely perceptible): 1
- Slight edema (edges of area well defined by definite raising): 2
- Moderate edema (raised approximately 1 mm): 3
- Severe edema (raised more than 1 mm and extending beyond area of exposure): 4

Maximum possible edema score 4
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Remarks:
The primary irritation index is the mean value of the sum of the scores for erythema and edema from all 6 rabbits 24 and 72 hours after initiation of treatment. The maximum possible score is 8.
Time point:
other: 24-72 hours
Score:
3
Max. score:
8
Reversibility:
not fully reversible within: 3 days
Remarks on result:
probability of moderate irritation
Irritant / corrosive response data:
The primary skin irritation index in this experiment was established to be 3.0. The test material caused therefore a moderate skin irritation to rabbits.

Skin irritation in individual rabbits

    24 hours after application     72 hours after application    
  Animal N° sex Skin reaction   Intact skin Scarified skin  Intact skin  Scarified skin 
 1 male Erythema 0
 1 male Edema 0
 2 male Erythema 2
 2 male Edema 2
 3 male Erythema 0
 3 female Edema 2
 4 female Erythema 0
 4 female Edema 2
 5 female Erythema 0
 5 female Edema 1
 6 female Erythema 2
 6 female Edema 1

 

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
The primary irritation index, a measure of the acute irritation to the rabbit skin was 3.0. This indicates that the test material is a moderate irritant to the rabbit skin.
Executive summary:

The skin irritation potential of the test item was determined according to the procedure used was the patch test technique described in the "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959), the US Association of Food and Drug Officials (AFDO).

The test was performed on 3 male and 3 female rabbits of the Himalayan breed.

Two days before treatment the entire back and the flank of the rabbits were shaved and immediately before treatment the shaven skin on the left side was slightly scarified.

For treatment the test material was applied to the prepared abraded and non abraded skin, in quantities of 0.5 ml.

The patches were covered with an impermeable material (occlusive application) for 24 hours.

The skin irritation was scored immediately and 48 hours after removal of the dressings (24 and 72 hours after initiation of treatment).

The primary skin irritation index in this experiment was established to be 3.0. The test material caused therefore a moderate skin irritation to rabbits.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
other: The procedure used was the test for eye irritants described in the "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959), the US Association of Food and Drug Officials (AFDO).
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
not applicable
GLP compliance:
no
Species:
rabbit
Strain:
Himalayan
Details on test animals or tissues and environmental conditions:
The test was performed on 3 male and 3 female rabbits of the Himalayan breed weighing 1.5 to 2 kg. The animals were housed individually in metal cages, were kept at a constant room temperature of 22 + 1° C, at a relative humidity of 55 + 5 % and on a 10 hour light cycle day.
The animals had ad libitum access to Standard rabbit food - NAFAG, Gossau SG - and water. Prior to treatment they were adapted to our laboratories for a minimum of 4 days. Only rabbits with normal ophthalmip findings were used for these tests.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
0.1 mL was inserted into the conjunctival sac of the left eye.
Duration of treatment / exposure:
One single application
Observation period (in vivo):
7 days
Duration of post- treatment incubation (in vitro):
Not applicable
Number of animals or in vitro replicates:
6 rabbits
Details on study design:
The test material in an amount of 0.1 ml was inserted into the conjunctival sac of the left eye of the 6 rabbits and the lids were gently closed for one second.
The right eye was not treated and served as an untreated control. In 3 of the 6 rabbits approximately 30 seconds after treatment the treated eye was flushed with 10 ml of lukewarm water. The eye irritation was appraised with a slit-lamp on day 1, 2, 3, 4 and 7 and was scored for each individual rabbit as outlined in table 1.
The primary eye irritation index represents the sum of the mean values for cornea, iris and conjunctivae. This procedure was carried out separately for unrinsed and rinsed eyes respectively.

SCORING SYSTEM: Table 1

Score for eye irritation i n rabbits (Table 1)
CORNEA:
A) Opacity and degree of density (most dense area scored)
- No opacity: 0
- Scattered or diffuse area, details of iris clearly visible: 1
- Easily discernible translucent areas, details of iris slightly obscured: 2
- Opalescent areas, no details of iris visible, size of pupil barely discernible: 3
- Opaque, iris invisible: 4

B) Area of cornea involved
- 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

A x B x 5 Maximum possible score = 80


IRIS;
A) Values
- Normal: 0
- Folds above normal, congestion, swelling, circumcornea injection (any or all 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

A x 5 Maximum possible score = 10



CONJUNCTIVAE;
A) Redness (refers to palpebral and bulbar conjunctivae excluding cornea and iris)
- Vessels normal: 0
- Vessels definitely injected above normal: 1
- More diffuse, deeper crimson red, individual vessels not easily discernible: 2
- Diffuse beefy red: 3

B) Chemosis
- No swelling: 0
- Any swelling above normal (includes nictitating membrane): 1
- Obvious swelling with partial eversion of lids: 2
- Swelling with lids about half closed: 3
- Swelling with lids about half closed to completely closed: 4

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

(A + B + C) x 2 Maximum possible score = 20



Irritation parameter:
cornea opacity score
Remarks:
eyes not rinsed after treatment.
Basis:
mean
Remarks:
Rabbit 1, 2 and 3
Time point:
other: 1-7 days
Score:
15.7
Max. score:
80
Reversibility:
not fully reversible within: 7 days
Irritation parameter:
cornea opacity score
Remarks:
Eyes rinsed 30 sec. after treatment
Basis:
mean
Remarks:
Rabbit 4, 5 and 6
Time point:
other: 1-7 days
Score:
2.7
Max. score:
80
Reversibility:
fully reversible within: 4 days
Irritation parameter:
iris score
Remarks:
eyes not rinsed after treatment
Basis:
mean
Remarks:
Rabbit 1, 2 and 3
Time point:
other: 1-7 days
Score:
0
Max. score:
10
Irritation parameter:
iris score
Remarks:
Eyes rinsed 30 sec. after treatment.
Basis:
mean
Remarks:
Rabbit 4,5 and 6
Time point:
other: 1- 7 days
Score:
0
Max. score:
10
Irritation parameter:
conjunctivae score
Remarks:
Eyes not rinsed after treatment
Basis:
mean
Remarks:
Rabbit 1, 2 and 3
Time point:
other: 1-7 days
Score:
9.5
Max. score:
20
Reversibility:
not fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Remarks:
Eyes rinsed 30 sec. after treatment
Basis:
mean
Remarks:
Rabbit 4, 5 and 6
Time point:
other: 1-7 days
Score:
1.7
Max. score:
20
Reversibility:
fully reversible within: 2 days
Irritant / corrosive response data:
In the present experiment the iritaion index was established as 9.2 for cornea, 0 for Iris and 5.6 for the conjunctivae in not rinsed eyes after treatment.
Rinsing the eyes following instillation was of very good effect.

R=6 (R: 4.1-10) very good effect

Effect of rinsing:
R = unrinsed eyes / rinsed eyes = 8.4 / 1.4 = 6

Unrinsed eyes: (15.7 + 0 + 9.5) / 3 = 8.4
Rinsed eyes: (2.7 + 0 + 1.7) / 3 = 1.4

Eye irritation in individual rabbits with eyes not rinsed after treatment.

         Rabbit 1 (male) Rabbit 2 (male)        Rabbit 3 (male)       
 Days after Application Cornea  Iris  Conjunctivae  Cornea  Iris  Conjuctivae  Cornea  Iris  Conjunctivae 
 1  20  0  12  20  0  12  10  0  8
 2  20  14  20  0  12  10  0  6
 3  20  0  14  20  0  12  10  0  6
 4  20  0  12  20  0  10 10   0  6
 7  20  0  10  10  0  8  5  0  0

- Cornea: 15.7

- Iris: 0

- Conjunctivae: 9.5

       Rabbit 4 (female) Rabbit 5 (female)        Rabbit 6 (female)       
 Days after Application Cornea  Iris  Conjunctivae  Cornea  Iris  Conjuctivae  Cornea  Iris  Conjunctivae 
 1 5 0 8 5 0 6 5 0 6
 2 5 0 0 5 0 0 5 0 6
 3 0 0 0 5 0 0 0 0 0
 4 0 0 0 5 0 0 0 0 0
 7 0 0 0 0 0 0 0 0 0

- Cornea: 2.7

- Iris: 0

- Conjunctivae: 1.7

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Under the conditions of this experiment the test material was found to be classified as Category 2A (irrittaing to eyes) based on GHS criteria.
Rinsing the eyes following instillation was of very good effect.
Executive summary:

The test was performed to assess the irritation potential of the test item on in vivo rabbit eyes.

The test was done on 3 male and 3 female rabbits of the Himalayan breed.

The test material in an amount of 0.1 ML was inserted into the conjunctival sac of the left eye of the 6 rabbits and the lids were gently closed for one second.

The right eye was not treated and served as an untreated control. In 3 of the 6 rabbits approximately 30 seconds after treatemnt the treated eyes was flushed with 10 mL of lukewarm water.

In the present experiment the iritaion index was established as 9.2 for cornea, 0 for Iris and 5.6 for the conjunctivae in not rinsed eyes after treatment.

Rinsing the eyes following instillation was of very good effect.

R=6 (R: 4.1-10) very good effect

Effect of rinsing:

R = unrinsed eyes / rinsed eyes = 8.4 / 1.4 = 6

Under the conditions of this experiment the test material was found to be classified as Category 2A (irrittating to eyes) based on GHS criteria.

Rinsing the eyes following instillation was of very good effect.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

One skin irritation study was performed to assess potential irritant of the test item.

The test was performed on 3 male and 3 female rabbits of the Himalayan breed.

Two days before treatment the entire back and the flank of the rabbits were shaved and immediately before treatment the shaven skin on the left side was slightly scarified.

For treatment the test material was applied to the prepared abraded and non abraded skin, in quantities of 0.5 ml.

The patches were covered with an impermeable material (occlusive application) for 24 hours.

The skin irritation was scored immediately and 48 hours after removal of the dressings (24 and 72 hours after initiation of treatment).

The primary skin irritation index in this experiment was established to be 3.0. The test material caused therefore a moderate skin irritation to rabbits.

One eye irritation test was also performed to assess the irritation potential of the test item on in vivo rabbit eyes.

The test was done on 3 male and 3 female rabbits of the Himalayan breed.

The test material in an amount of 0.1 ML was inserted into the conjunctival sac of the left eye of the 6 rabbits and the lids were gently closed for one second.

The right eye was not treated and served as an untreated control. In 3 of the 6 rabbits approximately 30 seconds after treatemnt the treated eyes was flushed with 10 mL of lukewarm water.

In the present experiment the iritaion index was established as 9.2 for cornea, 0 for Iris and 5.6 for the conjunctivae in not rinsed eyes after treatment.

Rinsing the eyes following instillation was of very good effect.

Under the conditions of this experiment the test material was found to be classified as Category 2 (irrittaing to eyes) based on GHS criteria.

Rinsing the eyes following instillation was of very good effect.

Justification for classification or non-classification

Based on the above stated assessment of the skin irritation potential of bis(2-3-epoxypropyl)cyclohex-4-ene-1-2-dicarboxylate the substance does have to be classified as skin irritating category 2 according to Council Directive 2001/59/EC and according to CLP (Regulation (EC) No 1272/2008 Of the European parliament and of the Council.

 

Based on the above assessment of the eye irritation potential of bis(2-3-epoxypropyl)cyclohex-4-ene-1-2-dicarboxylate the substance does have to be classified as eye irritating Category 2 according to Council Directive 2001/59/EC and according to CLP (Regulation (EC) No 1272/2008 Of the European parliament and of the Council.

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