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Administrative data

Description of key information

In vivo skin irritation/corrosion test (OECD 404, GLP compliant) - positive result

In vivo eye irritation/corrosion test (OECD 405, GLP compliant) - negative result

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:
Sept - Oct 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr K. Thomae GMBH, Biberach
- Age at study initiation: Young Adults
- Weight at study initiation: 3.5kg - 3.97kg
Animal 1: 3.50kg (Male)
Animal 2: 3.95 kg (Male)
Animal 3: 3.97kg (Male)
- Housing: 1 animal per cage. Stainless steel wire mesh. Floor area 3000cm2. No bedding in the cages, sawdust in waste trays.
- Diet: Kliba-Labordiaet, Klingentalmuehle AG, Switzerland. About 350g per animal per day.
- Water: About 250mL tap water per animal per day
- Acclimation period: At least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.5ml applied in a patch 2.5cm x 2.5cm. The substance was heated to 90C and cooled to body temperature before application,


Duration of treatment / exposure:
4 hours
Observation period:
15 days. After the 4 hour exposure period, the test substance was removed with a Polyethylene glycol/water solution. Oberservations were made at the following intervals after removal of the patch:
1 hour
24 hours
48 hours
72 hours
8 days
15 days
A check for dead animals was made at least once a day.

Number of animals:
3
Details on study design:
TEST SITE
- Area of exposure:
Upper third of the back or flanks
- 0.5mls of substance, covering an area 2.5cm x 2.5cm
- Type of wrap: Semiocclusive; Idealbinde.

REMOVAL OF TEST SUBSTANCE
- Washing:
With a Polyethylene Glycol (Lutrol E400) and water mix (2:1)
- Time after start of exposure:
4 hours

OBSERVATION TIME POINTS
1 hour
24 hours
48 hours
72 hours
8 days
15 days

In Animal 1, a cross incision of the treated skin was peformed after the animal was killed at study termination. Additional histopathological examination was performed.
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
2.7
Max. score:
4
Reversibility:
not reversible
Remarks:
within 15 days
Remarks on result:
positive indication of irritation
Irritation parameter:
erythema score
Remarks on result:
not determinable
Remarks:
Could not be read because of staining due to the colour of the test substance

Individual observations taken for each animal are tabulated below:

Time of observation

 Animal number

 

Erythema score

Edema score

 

Comments on symptoms

 

1 hour

 

1

-

3

Index for Erythema could not be read because of staining due to the colour of the test substance

2

-

2

3

-

1

24 hours

1

-

4

Index for Erythema could not be read because of staining due to the colour of the test substance

2

-

4

Index for Erythema could not be read because of staining due to the colour of the test substance. Index for Erythema of the surrounding area: 3

3

-

2

48 hours

1

-

3

Index for Erythema could not be read because of staining due to the colour of the test substance. Index for Erythema of the surrounding area: 2

2

-

3

3

-

2

72 hours

1

-

3

Superficial scabbing. Index for Erythema could not be read because of staining due to the colour of the test substance. Index for Erythema of the surrounding area: 2

2

-

2

Superficial scabbing. Index for Erythema could not be read because of staining due to the colour of the test substance. Index for Erythema of the surrounding area: 1

3

-

1

Index for Erythema could not be read because of staining due to the colour of the test substance. Index for Erythema of the surrounding area: 1

8 days

1

0

2

Superficial scabbing. Application area reddish discoloration.

2

-

1

Superficial scabbing. Index for Erythema could not be read because of staining due to the colour of the test substance

3

-

1

15 days

1

0

3

Gross Examination of the skin.

2

-

1

Index for Erythema could not be read because of staining due to the colour of the test substance

3

-

1

Superficial scabbing. Index for Erythema could not be read because of staining due to the colour of the test substance

Mean

1

-

3.3

 

2

-

3.0

 

3

-

1.7

 

Overall Mean

 

2.7

 

 

 

Note for the calculation of the mean, only the results at 24, 48 and 72 hours are used. 

PATHOLOGICAL-ANATOMICAL EVALUATION:

Skin, Left Flank: In the area of application surface raised, palpatory slight thickening of tissue.

HISTO-PATHOLOGICAL EXAMINATION:

Skin, Left Flank: Minimal Infiltration with neutrophile Granulocyte.

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
The test substance gave a mean Edema score of 2.7 and based on the observations in this test should be classified according to EU criteria as Skin Irritant Category 2.
Executive summary:

The skin irritation potential of the test material was evaluated in this study with New Zealand White rabbits. One group of three rabbits received a volume of 0.5 ml of the test substance applied in a patch 2.5cm by 2.5cm. on the upper third of the back or the flanks. The fur was removed by clipping. The material was first heated to 90C, then cooled to body temperature for application. Following the application, each site was covered with a semiocclusive dressing. Four hours post-dose, the dressings were removed. The exposure sites were cleaned to remove as much non-absorbed test article as possible. The exposure sites were examined and scored separately for both erythema and edema at the following time periods after removal of the patch: 1h, 24h, 48h, 72h, 8d and 15d.

The evaluation of erythema was not possible, because the test substance caused red coloration of skin. The mean edema score was 2.7 and the findings were not reversible after 15 days. Pathological-Anatomical examination of 1 animal in the area of application showed surface raised, palpatory slight thickening of tissue. The Histopatholoogical examination showed minimal infiltration with neutrophil granulocyte.

Based on these observations and the edema score, the substance should be classified according to EU criteria as Skin Irritant Category 2.

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:
Nov 1997 - Mar 1998
Reliability:
1 (reliable without restriction)
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
1987
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source:
Dr K. Thomae GMBH, BIBERACH
- Age at study initiation:
Young adult
- Weight at study initiation: 3.77kg - 3.96kg
Animal 1: 3.77kg (Male)
Animal 2: 3.96kg (Male)
Animal 3: 3.92kg (Male)
Animal 4: 3.78kg (Male)
- Housing: 1 animal per cage. Stainless steel wire mesh. Floor area 3000cm2. No bedding in the cages, sawdust in waste trays.
- Diet: Kliba-Labordiaet, Klingentalmuehle AG, Switzerland. About 130g per animal per day.
- Water: About 250mL tap water per animal per day
- Acclimation period: At least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Vehicle:
unchanged (no vehicle)
Controls:
other:
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.1ml
The material was first heated to 90C then cooled to body temperature before application.
Duration of treatment / exposure:
24 hours (24 hours after application the substance was washed out with tap water)
Observation period (in vivo):
1 hour
24 hour (the 24 hour reading was taken after the substance had been washed out.
48 hour
72 hour
8 days (Animals 2 and 3 only)
15 days (Animals 2 and 3 only)
21 days (Animals 2 and 3 only)
28 days (Animals 2 and 3 only)
Number of animals or in vitro replicates:
4 animals in total
1 animal in an intitial step.
As no further findings were noted, a further 2 confirmatory animals. Some symptoms were observed in one of these animals and it was unclear if they were due to the test substance.
Therefore, to clarify the results, a 4th animal was then tested.

Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done):
Yes
- Time after start of exposure:
24 hours

Initially 3 animals had the test substance applied. As the irritant potential of the substance was unknown, 1 animal was tested as a first step. As no severe findings were noted, another 2 rabbits were tested. To clarify the results, a further animal was tested.
Animal 1 initially application date 03-Nov-97
Animals 2 and 3 application date: 10-Nov-97
The effects were reversible in 2 animals, In one animal, opacity grade 1 over an area of less than a quarter was observed from 24 hours to 28 days. As no effects on the cornea were observed in the other 2 animals, and the grade or size of the observed corneal opacity did not change during the observation period, it was assumed that it was not related to the test substance. Therefore another animal was tested and the animal with corneal opacity was excluded from the evaluation of eye irritancy properites of the test substance.
Animal 4 application date: 12-Jan 1998.

The findings which were noted in animal 4 were reversible within 48 hours after application, thus the observation period for this animal was terminated after 72 hours.
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:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
0.5
Max. score:
2
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0.2
Max. score:
1
Remarks on result:
no indication of irritation

The mean scores were calculated based on the readings at 24 hour, 48 hours and 72 hours. The full individual readings are tabulated below. Animal 2 was excluded from the mean calculations as it was determined that the effects seen were not due to the test substance.

Tabulation of irritant response data for each individual animal at each observation time as well as calculation of the means.

 

Time point of reading

Animal

Cornea

IRIS

CONJUCTIVA

SYMPTOMS (see key below)

OP

AR

RED

SW

DI

 1 hour

01

-

-

-

-

1

2

A

02

-

-

-

-

1

2

A

03

-

-

-

-

1

2

A

04

-

-

-

-

1

1

A, B

24 hours

01

0

0

0

1

1

1

B

02

1

1

0

2

1

1

B

03

0

0

0

2

0

1

B

04

0

0

0

0

1

1

B

48 hours

01

0

0

0

0

0

0

B

02

1

1

0

2

1

0

B

03

0

0

0

1

0

0

B

04

0

0

0

0

0

0

B

72 hours

01

0

0

0

0

0

0

SD, B

02

1

1

0

1

0

0

B

03

0

0

0

1

0

0

B

04

0

0

0

0

0

0

SD, B

8 days

02

1

1

0

0

0

0

B

03

0

0

0

0

0

0

B

15 days

02

1

1

0

0

0

0

B

03

0

0

0

0

0

0

B

21 days

02

1

1

0

0

0

0

B

03

0

0

0

0

0

0

B

28 days

02

1

1

0

0

0

0

B

03

0

0

0

0

0

0

B

Mean (24h, 48h 72h results)

01

0.0

 

0.0

0.3

0.3

 

 

02

1.0

 

0.0

1.7

0.7

 

Animal excluded from evaluation

03

0.0

 

0.0

1.3

0.0

 

 

04

0.0

 

0.0

0.0

0.3

 

 

Overall Mean

 

0.3

 

0.0

0.8

0.3

 

 

Overall mean excluding animal 2

 

0.0

 

0.0

0.5

0.2

 

 

 

Symptoms Key

A: Index for corneal opacity, area, iris and conjunctivae redness could not be read because of staining due to the colour of the test substance. 

B: Fur of the head reddish discoloured.

SD: Study discontinued because the animal was free of symptoms.

 

 

 

 

 

Interpretation of results:
GHS criteria not met
Remarks:
Not irritating
Conclusions:
The test substance is not classified as irritating to eyes according to EU CLP criteria.
Executive summary:

The test material was assessed for eye irritation/corrosion according to the Method B.5 Acute Toxicity: Eye Irritation/Corrosion. Council Regulation (EC) No.440/2008, and OECD Test guideline 405.

The potential of the test substance to cause damage to the conjuntiva, iris or cornea was assessed in 4 white New Zealand rabbits, subjected to a singular ocular application of 0.1ml of the test susbstance. The eyes were examined at 1 hour, 24 hours, 46 hours and 72 hours. For 2 of the animals examinations were also carried out at 8, 15, 21 and 28 days. One animal was subsequently excluded from the study due to eye effects not attributable to the test substance. The avergage score (24 to 74 hours) for irritation was calculated to be 0.0 for corneal opacity, 0.0 for Iris, 0.5 for Conjunctival redness and 0.2 for Chemosis.

The individual animal mean scores for the 24, 48 and 72 hr time-point indicated that this material would not be classified under EU regulations.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

IN VIVO SKIN IRRITATION:

The skin irritation potential of the test material was evaluated in this study with New Zealand White rabbits. One group of three rabbits received a volume of 0.5 ml of the test substance applied in a patch 2.5cm by 2.5cm. on the upper third of the back or the flanks. The fur was removed by clipping. The material was first heated to 90C, then cooled to body temperature for application. Following the application, each site was covered with a semiocclusive dressing. Four hours post-dose, the dressings were removed. The exposure sites were cleaned to remove as much non-absorbed test article as possible. The exposure sites were examined and scored separately for both erythema and edema at the following time periods after removal of the patch: 1h, 24h, 48h, 72h, 8d and 15d.

The evaluation of erythema was not possible, because the test substance caused red coloration of skin. The mean edema score was 2.7 and the findings were not reversible after 15 days. Pathological-Anatomical examination of 1 animal in the area of application showed surface raised, palpatory slight thickening of tissue. The Histopatholoogical examination showed minimal infiltration with neutrophil granulocyte.

Based on these observations and the edema score, the substance should be classified according to EU criteria as Skin Irritant Category 2.

IN VIVO EYE IRRITATION

The potential of the test substance to cause damage to the conjunctiva, iris or cornea was assessed in 4 white New Zealand rabbits, subjected to a singular ocular application of 0.1ml of the test subsstance. The avergage score (24 to 74 hours) for irritation was calculated to be 0.0 for corneal opacity, 0.0 for Iris, 0.5 for Conjunctival redness and 0.2 for Chemosis. First 1 animal was tested and as no severe findings were noted a further 2 animals were tested. In one of these rabbits grade 1 opacity was observed from 24 hours until 28 days. As no corneal effects were seen in the other 2 animals, and the grade and size of the observed corneal opacity did not change, it was assumed that this could not be attributed to the test substance. This animal was therefore excluded from the calculation of the mean and a fourth confirmatory animal was tested.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to eye irritation.

 

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does require classification with respect to skin irritation as Category 2 (H315; Causes skin irritation).