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Description of key information

- Skin irritation: not irritating, male, New-Zealand White rabbit, OECD 404, Matting 2015


- Eye irritation: not irritating, male, New-Zealand White rabbit, OECD 405, Matting 2015

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:
14 Apr 2015 to 19 Apr 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This in vivo study was conducted to meet the data requirements of regulations not related to REACH in non-EEA countries.
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
April 2002
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2500 (Acute Dermal Irritation)
Version / remarks:
August 1998
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
May 2008
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS:
- Age at study initiation: 12 weeks
- Weight at study initiation: 3113 – 3282 g
- Housing: Individual caging
- Diet: ad libitum.
- Water: Tap water, from an automatic system, ad libitum
- Acclimatisation period: 13 and 15 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 – 22.4
- Humidity (%): 28 – 53
- Air changes (per hour): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 Apr 2015 To: 19 Apr 2015
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
water
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.5 g
Duration of treatment / exposure:
4 hours
Observation period:
72 hours
Number of animals:
3
Details on study design:
STUDY DESIGN
In a primary skin irritation study, the test substance was administered at 0.5 g / animal, the dose specified in the test guidelines for a solid test substance. Initially, a single animal was treated. As neither a corrosive effect nor a severe irritant effect were observed after the 24-hour observation, the test was completed using the 2 remaining animals with an exposure period of 4 hours.
The viability/mortality was recorded daily from the day of application of the animals to the termination of test.
Clinical signs were recorded daily and body weights were recorded on the day of application and at termination of observation.
The animals were checked daily for signs of systemic toxicity.

TEST SITE
- Area of exposure: Approximately 24 hours prior to the test the hair was clipped from the back and flanks of the animals with an electric clipper, exposing an area of approximately 100 cm2 (10 cm x 10 cm). 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.
- Type of wrap: On the day of treatment, 0.5 g of test substance was placed on a surgical gauze pad (ca. 2.5 cm x 2.5 cm) and sufficient water was added to dampen the material to ensure good contact with the skin. This gauze pad was applied to the intact skin of the clipped area and was kept in contact with the skin by a patch with a surrounding adhesive hypoallergenic plaster. The entire trunk of the animals was then wrapped with plastic wrap held in place with an elastic stocking.

REMOVAL OF TEST SUBSTANCE
- Duration of treatment: 4 hours
- Washing: The dressing was then removed and the skin was flushed with lukewarm tap water to clean the application site.

OBSERVATION TIME POINTS
- Approximately 1, 24, 48 and 72 hours after the removal of the dressing, gauze patch and test substance.  

SCORING SYSTEM:
- Method of calculation: Draize scoring system. The primary irritation index was calculated by totalling the mean cumulative scores at 24, 48 and 72 hours for all animals and then dividing by the number of data points.
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
animal: #1, #2, #3
Time point:
24/48/72 h
Score:
0
Max. score:
5
Irritation parameter:
erythema score
Basis:
animal: #1, #2, #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal: #1, #2, #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
No local dermal signs were observed in the treated animals throughout the study.
Other effects:
CLINICAL OBSERVATIONS
No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.

DURATION OF THE IN-LIFE PHASE
As no clinical signs were observed at 72 hours after patch removal, the study was terminated after the 72 hours observation.

BODY WEIGHT
The body weights of all rabbits were considered to be within the normal range of variability.
Interpretation of results:
GHS criteria not met
Conclusions:
The application of the test substance did not result in any signs of skin irritation.
Under the conditions of this study, the test substance is considered to be "not irritating" to the rabbit skin according to the Draize classification criteria.
Executive summary:

The primary skin irritation potential of the test substance was investigated according to the GLP compliant OECD 404 (2002), OPPTS 870.2500 (1998) and EC No 440/2008, B.4 (2008). Young adult New Zealand White rabbits (3 males) were dermally exposed to 0.5 g of test substance, applied to the intact shaved skin under a semi-occlusive dressing, for 4 hours. Skin reactions were scored at 1, 24, 48 and 72 hours after removal of the dressings according to the Draize Scoring System. The Primary Irritation Index (PII) was calculated.

No local dermal signs were observed in the treated animals throughout the study. No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred. As no clinical signs were observed at 72 hours after patch removal, the study was terminated after 72 hours observation. The body weights of all rabbits were considered to be within the normal range of variability. The application of the test substance  did not result in any signs of skin irritation.

Under the conditions of this study, the test substance is considered to be not irritating to the rabbit skin according to the Draize classification criteria.

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:
weight of evidence
Study period:
14 Apr 2015 to 19 Apr 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This in vivo study was conducted to meet the data requirements of regulations not related to REACH in non-EEA countries.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
October 2012
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Version / remarks:
August 1998
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
May 2008
Qualifier:
according to guideline
Guideline:
other: Directive 2004/73/EC B.5 (L 152)
Version / remarks:
April 2004
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Age at study initiation: 12 weeks (young adult)
- Body weight at study initiation: 3081 – 3219 g
- Housing: Individual caging in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages.
- Diet: ad libitum
- Water: Tap water, from an automatic system, ad libitum
- Acclimatisation period: at least 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 – 22.4
- Humidity (%): 28 – 53
- Air changes (per hour): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 14 Apr 2015 End: 19 Apr 2015
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.1 g
Duration of treatment / exposure:
1 hour
Observation period (in vivo):
1, 24, 48 and 72 hours
Number of animals or in vitro replicates:
3 males
Details on study design:
PRE-STUDY EXAMINATION
Before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect. Additionally, to assess the presence of corneal damage, fluorescein staining was employed at least approximately 24 hours prior to instillation, using a hand-held slit-lamp. Only animals free of ocular damage were used.

CHRONOLOGY OF ANIMAL USE
Initially only one rabbit was treated with test substance. The local effects were non-irritant (all scores were zero) at 24 hours, then two further rabbits were treated with the test substance.

ANALGESIC AND ANAESTHETIC TREATMENT
Sixty minutes (60 ± 10 min) prior to test substance application, a systemic opiate analgesic was administered by subcutaneous injection under direct Veterinary supervision. Repeat injections were given on the first day as appropriate to maintain an adequate level of analgesia.
Five minutes (5 ± 1.5 min) prior to test substance application, a topical ocular anaesthetic was applied to each eye (including the control eye) to ensure direct comparison of any ocular observations.
Eight hours (8 to 9 hr) after test substance application, a systemic opiate analgesic and a nonsteroidal anti-inflammatory drug (NSAID) were administered by subcutaneous injection under direct Veterinary supervision.
The systemic opiate analgesic was again injected ~ 12 hours after the post-treatment analgesic and then every 12 hours, and NSAID injected every ~ 24 hours, until eye scores were zero.
- Systemic opiate analgesic: Buprenorphine 0.01 mg/kg.
- Topical ocular anaesthetic: Humacain (oxybuprocaine) one-two drops per eye.
- Nonsteroidal anti-inflammatory drug: Meloxicam 0.5 mg/kg

APPLICATION OF THE TEST SUBSTANCE
The test substance was placed in the conjunctival sac of the left eye of the animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for at least one second in order to prevent loss of the material. The untreated contralateral eye served as the control.

DURATION OF EXPOSURE
The treated eye in all animals was rinsed with physiological saline solution at the first observation time point at one hour after application as the test substance remained in the eye sac

CLINICAL OBSERVATIONS AND EVALUATION OF OCULAR IRRITATION
Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours in all animals after test material installation. Observations with fluorescein staining were made approximately 24 hours before treatment and then 24, 48 and 72 hours after treatment in all animals. The duration of the observation period was sufficient to identify reversibility or irreversibility of changes. Any clinical signs of toxicity or signs of ill-health during the study were recorded. All rabbits were examined for distress at least twice daily, with observations at least 6 hours apart. Clinical observations or signs of ill-health were recorded.

MEASUREMENT OF BODY WEIGHT
Individual body weight was recorded on the day of treatment and before euthanasia .

POST MORTEM INVESTIGATIONS
At the end of the observation period, animals were euthanised by intramuscular injections of ketamin 10 % (Ketamidor) and xylazin 2 % (Primazin) followed by intravenous pentobarbital sodium (Euthanimal 40 %) anaesthesia. Following confirmation of death, the carcasses were discarded without further examination.

SCORING AND ASSESSMENT OF LOCAL REACTION
The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (02 October 2012).
Irritation parameter:
cornea opacity score
Basis:
animal: #1, #2, #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal: #1, #2, #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal: #1, #2, #3
Time point:
24/48/72 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal: #1, #2, #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
- No Initial Pain Reaction/Pain Reaction (IPR/PR) was observed.
- Conjunctival redness (score 2) was seen in all rabbits and chemosis (score 1) was seen in one rabbit at 1 hour after treatment. The test substance remained in the eye sac at 1 hour observation in all animals. At 24, 48 and 72 hours after the application, no clinical signs and no conjunctival or corneal effects were observed in any animal.
- Fluorescein staining was negative in all animals during the experiment.
- The control eyes were symptom-free during the study.
Other effects:
BODY WEIGHT
The body weights of all rabbits were considered to be within the normal range of variability.

CLINICAL SIGNS
No clinical signs of systemic toxicity were observed in any animal in this study.

MORTALITY
No mortality occurred during this study.

Table 2 Individual Draize Scores and Individual Total Scores* for Ocular Irritation

Rabbit number and sex

 

Male 1

 

 

Male 2

 

 

Male 3

 

IPR

 

 

0

 

 

0

 

 

0

 

PR

0

0

0

0

0

0

0

0

0

0

0

0

Time after treatment

1

24

48

72

1

24

48

72

1

24

48

72

Hr

Hr

Hr

Hr

Hr

Hr

Hr

Hr

Hr

Hr

Hr

Hr

CORNEA

 

 

 

 

 

 

 

 

 

 

 

 

E = Degree of Opacity

0

0

0

0

0

0

0

0

0

0

0

0

F = Area of Cornea involved

0

0

0

0

0

0

0

0

0

0

0

0

* Score (E x F) x 5

0

0

0

0

0

0

0

0

0

0

0

0

IRIS

 

 

 

 

 

 

 

 

 

 

 

 

D

0

0

0

0

0

0

0

0

0

0

0

0

* Score (D x 5)

0

0

0

0

0

0

0

0

0

0

0

0

CONJUNCTIVAE

 

 

 

 

 

 

 

 

 

 

 

 

A = Redness

2

0

0

0

2

0

0

0

2

0

0

0

B = Chemosis

1

0

0

0

0

0

0

0

0

0

0

0

C = Discharge

0

0

0

0

0

0

0

0

0

0

0

0

* Score (A+B+C) x 2

6

0

0

0

4

0

0

0

4

0

0

0

* Total Score

6

0

0

0

4

0

0

0

4

0

0

0

Initial painreaction

PR:   Painreaction

Hr:     Hour(s)

* Kay J H and Calandra J C (1962)

Table 3 Individual Total Scores and Group Mean Scores for Ocular Irritation Calculated from the Draize Scores

Rabbit Number and Sex

* Individual Total Scores At:

1

24

48

72

Hour

Hours

Hours

Hours

Male 1

6

0

0

0

Male 2

4

0

0

0

Male 3

4

0

0

0

* Group Total

14

0

0

0

* Group Mean Score

 

4.67

 

0.00

 

0.00

 

0.00

*: Kay J H and Calandra J C (1962)

Table 4: Individual Body weights and Body weight Change

Rabbit Number and Sex

Individual Body weight (g)

Body weight Change (g)

Before treatment

At termination

Male 1

3219

3275

56

Male 2

3173

3235

62

Male 3

3081

3187

106

Interpretation of results:
GHS criteria not met
Conclusions:
The test substamce was graded as a minimal irritant (Class 3 on a 1 to 8 scale) to the rabbit eye according to the modified Kay and Calandra classification system. Although the authors consider the test substance to be a mild irritant, classification under CLP is not warranted.
Executive summary:

The primary eye irritation effect of the test substance was evaluated according to OECD 405 (2012), EPA OPPTS 870.2400 (1998), Directive 2004/73/EC B.5 (2004) and EC No 440/2008, B.5 (2008) and in compliance with GLP using 3 young adult male New Zealand White rabbits. The test substance was administered as an installation of a single dose of 0.1 g into the conjunctival sac of the left eye with the untreated right eye serving as control. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours after test material installation in all animals. Observations with fluorescein staining were made approximately 24 hours before treatment and then 24, 48 and 72 hours after the treatment in all animals. Rabbits were treated with analgesic and anaesthetic as per the regulatory guideline. Results obtained from these three animals were used to classify the test substance for irritation potential.

No Initial Pain Reaction/Pain reaction (IPR/PR) was observed. Conjunctival redness (score 2) was seen in all rabbits and chemosis (score 1) was seen in one rabbit at 1 hour after treatment. Test substance remained in the eye sac at 1 hour observation in all animals. At 24, 48 and 72 hours after the application, no clinical signs and no conjunctival or corneal effects were observed in any animal. Fluorescein staining was negative in all animals during the experiment. The control eye of animals was symptom-free during the study. No mortality occurred during the study. The body weights of all rabbits were considered to be within the normal range of variability.

The test substance was graded as a minimal irritant (Class 3 on a 1 to 8 scale) to the rabbit eye according to the modified Kay and Calandra classification system.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation


The primary skin irritation potential of the test substance was investigated under GLP to OECD TG 404. Three male young adult New Zealand White rabbits were dermally exposed to 0.5 g of test substance, applied to the intact shaved skin under a semi-occlusive dressing, for 4 hours. Skin reactions were scored at 1, 24, 48 and 72 hours after removal of the dressings according to the Draize Scoring System. The Primary Irritation Index (PII) was calculated. No local dermal signs were observed in the treated animals throughout the study. No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred. As no signs of dermal irritation or clinical signs were observed at 72 hours after patch removal, the study was terminated. The body weights of all rabbits were considered to be within the normal range of variability. The application of the test substance did not result in any signs of skin irritation. Under the conditions of this study, the test substance was considered to be not irritating to the rabbit skin.


Eye irritation


The primary eye irritation potential of the test substance was evaluated under GLP to OECD TG 405 (2012) using 3 young adult male New Zealand White rabbits. The test substance was instilled as a single dose of 0.1 g into the conjunctival sac of the left eye. The untreated right eye was serving as control. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours after test material installation in all animals. Observations with fluorescein staining were made approximately 24 hours before treatment and then 24, 48 and 72 hours after the treatment in all animals. Rabbits were treated with analgesic and anaesthetic as per the regulatory guideline. No initial pain reaction or pain reaction (IPR/PR) was observed. Conjunctival redness (score 2) was seen in all rabbits and chemosis (score 1) was seen in one rabbit at 1 hour after treatment. Test substance remained in the eye sac at 1 hour observation in all animals. At 24, 48 and 72 hours after the application, no clinical signs and no conjunctival or corneal effects were observed in any animal. Fluorescein staining was negative in all animals during the experiment. The control eye of animals was symptom-free during the study. No mortality occurred during the study. The body weights of all rabbits were considered to be within the normal range of variability.


The test substance was graded as a minimal irritant (class 3 on a 1 to 8 scale) to the rabbit eye according to the modified Kay and Calandra classification system.

Justification for classification or non-classification

Based on the available experimental data, classification for skin and eye irritation is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.