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Description of key information

Skin irritation: Not irritating (in vitro), OECD 439, EU Method B. 46, Kiss 2012c. 
Eye irritation: Not irritating (in vivo), OECD 405, EU Method B. 5 and EPA OPPTS 870. 2400, Török-Bathó 2012.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 March 2012 to 30 March 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.G
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Details on test animals or test system and environmental conditions:
EPISKIN-SM
Supplier: SkinEthic, France
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg
Duration of treatment / exposure:
15 ± 0.5 minutes
Number of animals:
Not applicable
Irritation / corrosion parameter:
% tissue viability
Value:
95
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects:
CELL VIABILITY
- Cell viability of treated disks was 95 %, see Table 1 for results.

VALIDITY OF THE TEST
The mean OD value of the three negative control tissues was 0.641. The positive control result showed 26 % viability. Each standard deviation value (SD) of the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

INDICATOR FOR POTENTIAL FALSE VIABILITY
> Possible direct MTT reduction with the test material: No colour change was observed after three hours of incubation of the test item in MTT solution. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
> Colouring potential of the test material: As the test material has an intrinsic colour, one additional chemical-treated tissue was used for the non specific OD evaluation. Mean OD (measured at 540 nm) of this tissue was determined as 0.025, Non Specific Colour % was calculated as 4 %. Therefore additional data calculation was not necessary.

Table 1: Optical Density (OD) and the Calculated % Viability

Substance

Optical Density (OD)

Viability (%)

Negative Control

1

0.639

100

2

0.607

95

3

0.676

105

Mean

0.641

100

Standard Deviation (SD)

5.00

Positive Control

1

0.150

23

2

0.191

30

3

0.155

24

Mean

0.165

26

Standard Deviation (SD)

3.79

Test Material

1

0.555

87

2

0.627

98

3

0.633

99

Mean

0.605

95

Standard Deviation (SD)

6.66

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the study, exposure to the test material resulted in a mean relative cell viability of 95 %. Since the cell viability was determined to be > 50 % of the negative control the test material was considered to be non-irritating.
Executive summary:

The potential for the test material to cause skin irritation was predicted in an in vitro study using the EPISKIN Model. The study was conducted under GLP conditions and in line with OECD 439 and EU Method B.46. The test material was applied topically to the surface of the skin for 15 minutes, which was terminated by rinsing with PBS 1 x solution (0.9 %). Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified. Positive and negative controls were run concurrently and tissue viability was expressed as a % relative to the negative control.

Under the conditions of the test, exposure to the test material resulted in a mean relative cell viability of 95 %. Since the cell viability was determined to be > 50% of the negative control the test material was considered to be non-irritating.

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:
26 April 2012 to 29 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
(the eyes of the test animals were washed 1 hour after administration of the test material).
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
yes
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
yes
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: Approximately 14 weeks old (adults).
- Weight at study initiation: Males ranged from 3251 to 3518 g.
- Housing: Individually in open wire cages.
- Diet: rabbit diet, ad libitum.
- Water: Municipal tap water, ad libitum.
- Acclimation period: 29 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): 15 to 20 per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light per day between 06:00 and 18:00 hours.

IN-LIFE DATES: From: 26 April To: 29 April
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g.
Duration of treatment / exposure:
1 hour
Observation period (in vivo):
72 hours.
Number of animals or in vitro replicates:
Three
Details on study design:
DOSING
- Procedure: An initial test was performed using one animal to assess the initial pain reaction. After consideration of the ocular responses produced in the first animal, one hour after the treatment of the first animal, two additional animals were treated.
- Administration: The test material was instilled into the conjunctival sac of the left eye. The eyelids were held closed for a few seconds to prevent the loss of the test item.

SCORING SYSTEM
- Initial pain reaction: Immediately after the administration of the test material, an assessment of the initial pain reaction was made according to the six point scale shown in Table 1.
- Ocular response: The eyes were examined at 1, 24, 48 and 72 hours after treatment. The duration of the observation period was sufficient to identify reversibility or irreversibility of changes. The eye irritation scores were evaluated according to the scoring system by Draize (1977) shown in Table 2.

OTHER OBSERVATIONS:
- Clinical: Any clinical signs of toxicity or signs of ill-health during the study were recorded.
- Bodyweight: Individual body weight was recorded at the beginning and end of the experiment.
- Necropsy: Performed at termination of the observation period.
Irritation parameter:
cornea opacity score
Remarks:
opacity
Basis:
animal #1
Time point:
other: Mean at 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Remarks:
opacity
Basis:
animal #2
Time point:
other: Mean at 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Remarks:
opacity
Basis:
animal #3
Time point:
other: Mean at 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #1
Time point:
other: Mean at 24, 48 and 72 hours
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #2
Time point:
other: Mean at 24, 48 and 72 hours
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #3
Time point:
other: Mean at 24, 48 and 72 hours
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Time point:
other: Mean at 24, 48 and 72 hours
Score:
0
Max. score:
3
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Time point:
other: Mean at 24, 48 and 72 hours
Score:
0
Max. score:
3
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #3
Time point:
other: Mean at 24, 48 and 72 hours
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: Mean at 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: Mean at 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
other: Mean at 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritant / corrosive response data:
- Initial Pain Reaction (IPR): A score of 2 was observed in all animals.
- One hour after the application: Conjunctival redness (score 1 or 2) and discharge (score 1 or 2) were seen in all rabbits. One animal showed conjuctival chemosis (score 1).
- At 24 hours after treatment: Conjunctival redness (score 1) was seen in one rabbit.
- At 48 and 72 hours after treatment: No signs of eye irritation were observed.
- Reversibility: As all signs of eye irritation had fully reversed the study was terminated after a period of 72 hours observation.
Other effects:
- Mortality: None observed during the study.
- Bodyweight: The body weight and body weight change were considered to be normal with no indication of a treatment related effect. Bodyweights ranged from 3420 to 3553 g at termination representing a weight gain of between 35 and 169 g.
- Clinical: There were no clinical signs observed that could be related to treatment.

Table 3: Individual Scores for Ocular Irritation at 1 Hour

Animal No.

Score of Irritation

IPR

Conjunctivae

Opacity of Cornea

Iris

Control Eye

Other Signs

R

CH

D

OD

OE

R

1

2

1

1

0

0

0

0

-

2

2

1

0

1

0

0

0

0

-

2

3

2

0

2

0

0

0

0

-

2

 

Table 4: Individual Scores for Ocular Irritation at 24 Hour

Animal No.

Score of Irritation

Conjunctivae

Opacity of Cornea

Iris

Control Eye

Other Signs

R

CH

D

OD

OE

R

1

0

0

0

0

0

0

0

-

2

0

0

0

0

0

0

0

-

3

1

0

0

0

0

0

0

-

 

Table 5: Individual Scores for Ocular Irritation at 48 Hour

Animal No.

Score of Irritation

Conjunctivae

Opacity of Cornea

Iris

Control Eye

Other Signs

R

CH

D

OD

OE

R

1

0

0

0

0

0

0

0

-

2

0

0

0

0

0

0

0

-

3

0

0

0

0

0

0

0

-

 

Table 6: Individual Scores for Ocular Irritation at 72 Hour

Animal No.

Score of Irritation

Conjunctivae

Opacity of Cornea

Iris

Control Eye

Other Signs

R

CH

D

OD

OE

R

1

0

0

0

0

0

0

0

-

2

0

0

0

0

0

0

0

-

3

0

0

0

0

0

0

0

-

 

R = redness, CH = chemosis; D = discharge; OD = opacity degree of density; OE = extent of opaque area; IPR = initial pain reaction.

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the test, the test material was determined to be non-irritating. Slight conjunctival redness and discharge were observed in all three animals one hour after administration, and one animal also showed slight chemosis. All ocular reactions were fully reversed by 48 hours post exposure. These reactions were slight and below the limit of classification.
Executive summary:

The potential for the test material to cause eye irritation was assessed in an in vivo irritation study in rabbits. The study was performed under GLP conditions in line with standardised guidelines OECD 405, EU Method B.5 and EPA OPPTS 870.2400. The test material was administered to three male New Zealand white rabbits, in a single 1 hour application. Rabbits were assessed over 72 hours for signs of eye irritation, clinical sign of toxicity, mortality and bodyweight gain. Application of the test material to the rabbit’s eye caused slight conjunctival redness and discharge in all three animals, one hour after administration. One animal also showed slight chemosis at one hour. These slight signs of irritation were fully reversed within 48 hours, and subsequently the study was terminated after 72 hours. Under the conditions of the study, the test material was determined to be non-irritating.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation:

Kiss (2012c) has been provided as the key study, where the potential for the test material to cause skin irritation was assessed in vitro using the EPISKIN Model. The study was performed under GLP and in accordance with OECD 439 and EU Method B. 46. The study was performed to a high standard and as such has been assigned a reliability score of 1 in line with the principles for assessing data quality defined in Klimisch (1997). Under the conditions of the test, exposure to the test material resulted in a mean relative cell viability of 95 %. Since the cell viability was determined to be > 50 % of the negative control the test material was considered to be non-irritating.

Eye Irritation:

Török-Bathó (2012) has been provided as the key study, where the capacity for the test material to cause eye irritation was assessed in vivo in a study with rabbits. The study was performed under GLP and in accordance with OECD 405, EU Method B. 5 and EPA OPPTS 870. 2400. The study was performed to a high standard and as such has been assigned a reliability score of 1 in line with the principles for assessing data quality defined in Klimisch (1997). Application of the test material to the rabbit’s eye caused slight conjunctivae redness and discharge in all three animals, one hour after administration. One animal also displayed slight chemosis at one hour. These slight signs of irritation were fully reversed within 48 hours, and subsequently the study was terminated after 72 hours. Under the conditions of the study, the test material was determined to be non-irritating.

Kiss (2012d) has been provided as supporting data, where the potential for the test material to cause corrosion or severe irritation was assessed in vitro in isolated chicken eyes. The study was performed to a high standard and as such has been assigned a reliability score of 1 in line with the principles for assessing data quality defined in Klimisch (1997). Under the conditions of the test, exposure to the test material resulted in an overall ICE class of 1 x I and 2 x II, corresponding to a classification of non-irritating. Adherence of the test material to the cornea was observed after the post-treatment rinse. The cornea surface was not cleared up to 240 min after the post-treatment rinse. Further testing is required for classification to assess the in vivo significance, of the adherence. In vivo eye lids will probably clear the surface, but abrasion may occur. This study suggests that the test material does not cause either corrosion or severe eye irritation and is in agreement with the findings presented in the in vivo study.


Justification for selection of skin irritation / corrosion endpoint:
The key study was performed in vitro, in line with GLP and standardised guidelines. It was assigned a reliability score of 1 in accordance with the criteria outlined in Klimisch (1997). It was therefore considered suitable to be the key study for this endpoint.

Justification for selection of eye irritation endpoint:
The key study was performed in vivo, in line with GLP and standardised guidelines. It was assigned a reliability score of 1 in accordance with the criteria outlined in Klimisch (1997). It was therefore considered suitable to be the key study for this endpoint.

Justification for classification or non-classification

Skin Irritation:

According to the criteria outlined in Regulation (EC) No. 1272/2008, the test material does not meet the criteria for classification as a skin irritant.

Eye Irritation:

According to the criteria outlined in Regulation (EC) No. 1272/2008, the test material does not meet the criteria for classification as an eye irritant.