Registration Dossier

Administrative data

Description of key information

In vivo skin irritation (OECD 404): Not irritating to rabbit skin

In vivo eye irritation (OECD 405): Not irritating to rabbit eyes

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:
Experimental starting date: 27 February 2008 and Experimental completion date: 06 March 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
- Animals: Young Adult New Zealand White Rabbit, SPF
- Rationale: Recognized by international guidelines as a recommended test system.
- Number of Animals per Test: 3 (Animals of both sexes were used)
- Age when treated: 17 weeks (male) and 13 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.
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
0.5 gram
Duration of treatment / exposure:
4 hours
Observation period:
72 hours
Number of animals:
3 animals of both sexes were used.
Details on study design:
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 clipped again.

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 TKK 30302 (XU 3560 / LMB 6452) was placed on a surgical gauze patch (ca. 2.5 cm x 2.5 cm). This gauze patch was applied to the intact skin of the clipped area. The patch was covered with a semi-occlusive dressing. 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.

SCORING SYSTEM:
COMMISSION DIRECTIVE 2004/73/EC, APRIL 29, 2004
Grading of Skin Reactions

ERYTHEMA AND ESCHAR FORMATION
- No erythema 0
- Very slight erythema 1
- Well-defined erythema 2
- Moderate to severe erythema 3
- Severe erythema (beef redness) or eschar
- formation (injuries in depth preventing erythema) reading 4

OEDEMA FORMATION
- No oedema 0
- Very slight oedema (barely perceptible) 1
- Slight oedema (edges of area well-defined by definite raising) 2
- Moderate oedema (edges raised approximately 1 mm) 3
- Severe oedema (raised more than 1 mm and extending beyond the area of exposure) 4
Irritation parameter:
overall irritation score
Basis:
mean
Remarks:
3 rabbits (1 male and 2 females)
Score:
0
Max. score:
0
Remarks on result:
other: No erythema and no edema were observed.
Irritant / corrosive response data:
Irritation
The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for oedema grades, separately.
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 individual mean score for erythema/eschar and oedema for each of the three animals was therefore 0.

Corrosion
Neither alterations of the treated skin were observed nor were corrosive effects evident on the skin.
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 skin was observed.
- Body weight: The body weights of all rabbits were considered to be within the normal range of variability.

Body weights in grams.

 Animal N° Sex  First day of acclimatization  Day of treatment  Last day of observation 
 75  male  2926  3039 3065 
 76  female  2430  2746 3046 
 77  female  2474  2600 2774 

Skin irritationscores-Individual values.

 Animal number Sex  Evaluation Interval*  Erythema  Edema  Scaling 
75   M   1 hour  0  0  0
 76  F   1 hour  0  0
 77  F   1 hour  0  0
 75  M   24 hours  0  0  0
 76  F   24 hours  0  0
 77  F   24 hours  0  0  0
 75  M  48 hours  0  0  0
 76  F  48 hours  0  0  0
 77  F  48 hours  0  0  0
 75  M  72 hours  0  0  0
 76  F  72 hours  0  0  0
 77  F  72 hours  0  0  0

*Examination were performed at the specified times after ramoval of the dressing.

SKIN IRRITATION SCORES – INDIVIDUAL MEAN VALUES AFTER 24, 48 AND 72 HOURS

 Animal number Sex  Erythema  Oedema 
 75  M  0.00  3  0.00  3
 76  F  0.00  3  0.00  3
 77  F  0.00  3  0.00  3

N= number of available data points.

SKIN IRRITATION SCORES – ASSESSMENT ACCORDING TO EC GUIDELINES

 Evaluated intervals  Erythema Oedema 
 24 hours   Not irritating   Not irritating
 48 hours   Not irritating   Not irritating
 72 hours   Not irritating   Not irritating

Individual Findings (for animal 75, 76 and 77).

After 1 hour:

Erythema: NO ABNORMAL FINDINGS NOTED

Oedema: NO ABNORMAL FINDINGS NOTED

Flaking: NO ABNORMAL FINDINGS NOTED

Staining: NO STAINING PRESENT

After 24 hours:

Erythema: NO ABNORMAL FINDINGS NOTED

Oedema: NO ABNORMAL FINDINGS NOTED

Flaking: NO ABNORMAL FINDINGS NOTED

Staining: NO STAINING PRESENT

After 48 hours:

Erythema: NO ABNORMAL FINDINGS NOTED

Oedema: NO ABNORMAL FINDINGS NOTED

Flaking: NO ABNORMAL FINDINGS NOTED

Staining: NO STAINING PRESENT

After 72 hours:

Erythema: NO ABNORMAL FINDINGS NOTED

Oedema: NO ABNORMAL FINDINGS NOTED

Flaking: NO ABNORMAL FINDINGS NOTED

Staining: NO STAINING PRESENT

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 2001), the test item is considered to be “not irritating” to rabbit skin.
Executive summary:

The primary skin irritation potential of the test item was investigated according to OECD test guideline no. 404. The test item was applied by topical semi-occlusive application of 0.5 g to the 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 mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for oedema grades, separately.

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 individual mean score for erythema/eschar and oedema for each of the three animals was therefore 0.

The test item caused no staining of the treated skin. No corrosive effects were noted on the treated skin of any animal at any of the measuring intervals and no clinical signs were observed.

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

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
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
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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.
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
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
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
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
Irritation parameter:
conjunctivae score
Basis:
animal #1
Remarks:
Animal number 75 (Male)
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
animal #2
Remarks:
Animal number 76 (Female)
Time point:
24/48/72 h
Score:
0
Max. score:
0
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.

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
 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  Iris  Redness  Chemosis 
 75  M  0.00  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

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Two tests were performed in vivo to assess the irritating potential of the test item to skin and rabbit eyes.

As in vivo test were already available for skin and eyes, no in vitro test need to be performed (Waiver).

The primary skin irritation potential of the test item was investigated by applying (during 4 hours) 0.5 g of the test item to the intact left flank of each of three young adult New Zealand White rabbits.

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 individual mean score for erythema/eschar and oedema for each of the three animals was therefore 0.

The test item caused no staining of the treated skin. No corrosive effects were noted on the treated skin of any animal at any of the measuring intervals and no clinical signs were observed.

In conclusion, the test item is considered to be “not irritating” to rabbit skin.

The primary eye irritation potential of the test item was investigated applying by instillation 0.1 g into the left eye of each of three young adult New Zealand White rabbits.

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.

In conclusion, the test item is considered to be “not irritating” to the rabbit eye.

The test item is neither irritant for skin and eye to rabbits

Justification for classification or non-classification

Based on the above stated assessment of the skin irritation of the test item, the substance does not have to be classified according to Council Directive 2001/59/EC and according to CLP (Regulation (EC) No 1272/2008 Of the European parliament and of the Council.

 

Base on the above assessment of the eye irritation potential of the test item, the substance does not have to be classified according to Council Directive 2001/59/EC and according to CLP (Regulation (EC) No 1272/2008 Of the European parliament and of the Council.