Registration Dossier

Administrative data

Description of key information

SKIN
Not irritating, OECD 404, EU Method B.4, Pooles (2009)
EYE
Not irritating, OECD 405, EU Method B.5, Pooles (2010)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 November 2009 to 13 November 2009
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 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Hillcrest, Belton, Loughborough, UK
- Age at study initiation: 12 - 20 weeks old
- Weight at study initiation: 2.17 - 2.43 kg
- Housing: Individually housed in suspended cages
- Diet: 2030 Teklad Global Rabbit diet (Harlan Teklad, Blackthorn, Bicester, Oxon, UK) available ad libitum
- Water: Mains drinking water available ad libitum
- Acclimation period: Minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): A minimum of 15 changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hour cycle
Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied : 0.5 g of test material sufficiently moistened in 0.5 mL of distilled water to achieve a paste.
Duration of treatment / exposure:
Animal were exposed for 4 hours
Observation period:
Animals were observed for 72 hours for skin reactions
Number of animals:
3 animals in total (1 animal used in the initial test, with a further 2 animals in the main test)
Details on study design:
TEST SITE
- Area of exposure: The test material was introduced under a 2.5 cm x 2.5 cm cotton gauze patch
- Type of wrap if used: The trunk of each rabbit was wrapped in an elasticated corset

REMOVAL OF TEST MATERIAL
- Washing: Any residual test material was removed by gentle swabbing with cotton wool soaked in distilled water
- Time after start of exposure: 4 hours post administration of test material

SCORING SYSTEM: Draize JH (1959) “Dermal Toxicity” In: Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Associations of Food and Drug Officials of the United States, Austin, Texas p.46-59
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Remarks:
of 3 animals
Time point:
other: 24 and 72 hours post dosing
Score:
0
Max. score:
8
Reversibility:
other: Not applicable, no effects observed during the study.
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Irritant / corrosive response data:
The scores for erythema and oedema at the 24 and 72-hour readings were totalled for the three test rabbits (12 values) and this total was divided by six to give the primary irritation index of the test material.

The test material produced a primary irritation index of 0.0.

Measurement of pH

The pH of the test material was determined prior to commencement of the study and found to be as follows:

Preparation

pH Measurement

immediately

after 10 minutes

after 20 minutes

 10 % w/w aqueous preparation of the test material

 8.9

 9.6

 9.6

Table 1: Individual Skin Reactions

Skin Reaction

Observation Time (Following Patch Removal)

Individual Scores – Rabbit Number and Sex

Total

68637 Male

68638 Male

68661 Male

Erythema/Eschar Formation

Immediately

0

0

0

(0)

1 Hour

0

0

0

(0)

24 Hours

0

0

0

0

48 Hours

0

0

0

(0)

72 Hours

0

0*

0*

0

Oedema Formation

Immediately

0

0

0

(0)

1 Hour

0

0

0

(0)

24 Hours

0

0

0

0

48 Hours

0

0

0

(0)

72 Hours

0

0*

0*

0

Sum of 24 and 72-hour readings (S): 0

Primary Irritation Index (S/6): 0/6 = 0.0

Classification: Non-Irritant

() = total values not included in calculating the primary irritation index

 

Table 2: Individual Bodyweights and Bodyweight Changes

Rabbit Number and Sex

Individual Bodyweight (kg)

Bodyweight Change (kg)

Day 0

Day 3

68637 Male

2.17

2.21

0.04

68638 Male

2.37

2.43

0.06

68661 Male

2.43

2.52

0.09

Interpretation of results:
not classified
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The test material produced a primary irritation index of 0.0 and was classified as a non-irritant to rabbit skin. No corrosive effects were noted.
Executive summary:

The skin irritation potential of the test material was investigated in vivo in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 404 and EU Method B.4.

During the study no signs of skin irritation were noted at any of the observation points. The test material was therefore concluded to be a non-irritant.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
7 July 2009 to 13 July 2009
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 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:
equivalent or similar to guideline
Guideline:
other: EU method B.46 (in vitro skin irritation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: EPISKIIN™ Reconstituted Human Epidermis model
Strain:
other: not applicable
Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: Positive and negative controls were included in the study
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg

VEHICLE
Test material was used as supplied
Duration of treatment / exposure:
15 minutes
Observation period:
42 hours
Number of animals:
The test was performed in triplicate
Details on study design:
APPLICATION OF TEST MATERIAL
- Area of exposure: The test material was applied topically to the reconstituted epidermis ensuring uniform coverage. The epidermis surface had been moistened with 5 µL of sterile distilled water to improve contact between the solid test material and the epidermis.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period each tissue was rinsed with a Phosphate Buffered Saline solution containing Ca2+ and Mg2+ before incubating for approximately 42 hours at 37 °C in 5% CO2 air
- Time after start of exposure: 15 minutes

SCORING SYSTEM:
At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.
At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm.
Irritation / corrosion parameter:
other: other: Relative tissue viability %
Value:
95 - 107.5
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes. Max. score: 100.0. Reversibility: other: not applicable. Remarks: Please refer to table 1 for tabulated results including control. (migrated information)
Other effects / acceptance of results:
The relative mean viability of the test material treated tissues was 101.8% after a 15-minute exposure.

QUANTITATIVE MTT ASSESSMENT (percentage tissue viability):For the test material, the relative mean tissue viabilities were compared to the mean of the negative control treated tissues (n = 3). The relative mean viabilities were calculated in the following way:

% Relative mean viability = (mean OD540 of test material/mean OD540 of negative control) x 100

The test material was found not to directly reduce MTT

Table 1: Mean OD540 values and % viabilitiesb for the negative control material, positive control material and test material

Material

OD540 of tissues

Mean OD540 of triplicate tissues

± SD of OD540

Relative individual tissue viability %

Relative mean % viability

± SD of % viability

Negative control material

0.826

 

0.756

 

0.061

109.3

 

100a

 

8.15

0.710

93.9

0.733

97.0

Positive control material

0.069

 

0.074

 

0.012

9.1

 

9.7

 

1.55

0.065

8.6

0.087

11.5

Test material

0.718

 

0.770

 

0.048

95.0

 

101.8

 

6.33

0.813

107.5

0.779

103.0

a The mean viability of the negative control tissues is set at 100%;

b Data are presented in the form of % viability (MTT reduction in the test material treated tissues relative to negative control tissues).

Table 2: Qualitative evaluation of tissue viability (MTT uptake visual evaluation)

Material

Tissue 1

Tissue 2

Tissue 3

Negative control Material

-

-

-

Positive Control Material

++

++

++

Test Material

-

-

-

MTT visual scoring scheme:

-    blue tissue (viable)

+ blue/white tissue (semi-viable)

++ tissue is completely white (dead)

Quality criteria

The quality criteria required for acceptance of results in the test were satisfied, i.e. positive control and negative control acceptance criteria.

Interpretation of results:
not irritating
Conclusions:
The test material was considered to be a non-irritant to the reconstituted human epidermis model EPISKIN™.
Executive summary:

The skin irritation potential of the test material was determined in vitro in a study which was conducted under GLP conditions and in accordance with the standardised guideline EU Method B.46.

Under the conditions of the study the test material was considered to be a non-irritant to the reconstituted human epidermis model EPISKIN™.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 October 2009 15 October 2009
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 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:
other: OECD 431 (In Vitro Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: Reconstituted Human Epidermal Model
Strain:
other: SkinEthic
Details on test animals or test system and environmental conditions:
- Supplier: SkinEthic Laboratories, Nice, France
- Date received : 13 October 2009
- Pre-incubation: Tissues were aseptically transferred into 6-well plates containing 1 mL maintenance medium at room temperature (Each tissue was inspected for any air bubble between the agarose gel and the tissue culture prior to transfer). The 6-well plated containing the tissues were polaced into an incubator overnight at 37 °C, 5 % CO2 in air.
- Medium: Produced in serum-free SkinEthic maintenance medium
Type of coverage:
other: Topical treatment
Preparation of test site:
other: Tissues on polycarbonate inserts
Vehicle:
unchanged (no vehicle)
Controls:
other: Positive and negative controls
Amount / concentration applied:
TEST MATERIAL
- Amount applied : 20 mg
- Preparation of test material : For the purpose of the test, the test material was ground before use.

CONTROL
- Negative control: 40 µL of sterile distilled water
- Positive control : 40 µL of 8.0 N potassium hydroxide (used as supplied)
Duration of treatment / exposure:
3 or 60 minutes
Observation period:
3 hours
Number of animals:
All tests were performed in duplicate
Details on study design:
TEST SITE
- Area of exposure: Tissue surface area
- Wetting : 20 µL of sterile distilled water was used for wetting the test material to ensure adequate contact with the tissue surface.

REMOVAL OF TEST SUBSTANCE
- Washing : At the end of each exposure period, each tissue was removed from the well of the treatment plate using forceps and rinsed using a wash bottle containing DPBS (Dulbecco's Phosphate Buffered Saline). Rinsing was achieved by filling and emptying each tissue insert with a constant soft stream of DPBS for approximately 40 seconds to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the tissue culture insert with absorbent paper.
- Time after start of exposure: 3 or 60 minutes.

SCORING SYSTEM: Corrosivity was determined by measuring the absorbency at 540 nm (OD 540) after treatment with MTT. The scoring system used is detailed in table 1.
Irritation / corrosion parameter:
other: other: % viability of tissues
Value:
97.8
Remarks on result:
other:
Remarks:
Basis: mean of two tissues. Time point: 3 minutes. Max. score: 100.0. Reversibility: other: Not applicable. (migrated information)
Irritation / corrosion parameter:
other: other: % viability of tissues
Value:
91.2
Remarks on result:
other:
Remarks:
Basis: mean of two tissues. Time point: 60 minutes. Max. score: 100.0. Reversibility: other: Not applicable. (migrated information)
Other effects / acceptance of results:
The OD540 for the 3 and 60 minute exposure of the SkinEthic model to FeMn slag were 1.216 and 1.260 respectively.

The relative mean viability of cell cultures compared to negative control tissues were calculated as follows:
Relative mean viability (%) = ((mean OD540 of test material)/(mean OD540 of negative control)) x 100

The test material was found not to directly reduce MTT.

Table 2: Mean OD540 Values and Percentage Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material

Exposure Time

Mean OD5401

Relative Mean Viability (%)

Negative control

3 Minute

 1.243

 100*

60 Minute

 1.381

 100*

Positive control

3 Minute

 0.058

 4.7

60 Minute

 0.048

 3.5

Test Material

3 Minute

 1.216

 97.8

60 Minute

 1.260

 91.2

1 Mean of SkinEthic tissues tested in duplicate

* Mean percentage viability of the negative control tissue is set at 100 %.

 

Interpretation of results:
other: No corrosion potential
Remarks:
Criteria used for interpretation of results: other: Based on the criteria set out in the OECD guideline
Conclusions:
The test material was considered not to have the potential to be corrosive in vivo.
Executive summary:

The skin corrosion potential of the test material was investigated in vitro, in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 431.

During the study 20 mg of test material was applied topically to reconstituted epidermis ensuring uniform coverage. At the end of the 3, or 60, minute exposure period each tissue was rinsed with Dulbecco's Phosphate Buffered Saline (DPBS) before being blotted and transferred to a well containing 300 µL of 0.5 mg/mL MTT solution. The tissues were then incubated at 37 °C, 5 % CO2 in air for 3 hours. At the end of the 3 hour MTT incubation period, the tissue cultures were blotted and transferred to pre-labelled 24-well plates for MTT extraction. The plates were left to stand overnight at room temperature to extract the reduced MTT (formazan dye) out of the tissue (ensuring the plate was covered to protect from light). At the end of the extraction period, each tissue was pierced and the extracted solution thoroughly mixed. For each tissue, triplicate 200 µL samples were taken.

The mean OD540 values of the triplicate wells, representing each SkinEthic RHE tissue, were automatically calculated by the plate reader. The mean OD540 values of the duplicate tissues were manually calculated. Each value had been corrected for blanks by the plate reader. For the test material, corrosivity potential was predicted from the relative mean tissue viabilities

Under the conditions of the study, the mean optical density of the test material at 540 nm following a 3 and a 60 minute exposure was 1.216 and 1.260, respectively. The mean % viability following a 3 and a 60 minute exposure was 97.8 and 91.2, respectively. Subsequently, the test material was concluded to be "non corrosive" to skin.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 November 2009 to 19 November 2009
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:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Limited, Hillcrest, Belton, Loughborough, UK
- Age at study initiation: 12 - 20 weeks old
- Weight at study initiation: 2.18 - 2.39 kg
- Housing: Animals were housed individually in suspended cages
- Diet: Free access to 2030 Teklad Global Rabbit diet (Harlan Teklad, Blackthorn, Bicester, Oxon, UK)
- Water: Free access to mains drinking water
- Acclimation period: A minimum of 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): At least 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour cycle.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied : 100 mg
Duration of treatment / exposure:
Up to 72 hours
Observation period (in vivo):
Animals were assessed up to 72 hours after exposure (assessments were made at 1, 24, 48 and 72 hours post treatment).
Number of animals or in vitro replicates:
1 animal was initially treated, two further animals were treated thereafter
Details on study design:
APPLICATION OF TEST MATERIAL:
Initially a single rabbit was treated with 100 mg of test material. The test material was administered into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment to prevent loss of test material and then released. The left eye served as a control and remained untreated. Immediately after administration of the test material and assessment of the initial pain reaction was made. After consideration of the ocular responses produced in the first treated animal, two additional animals were treated.

SCORING SYSTEM:
Assessment of ocular damage/irritation was made 1, 24, 48 and 72 hours post dosing using the scoring system from Draize JH (1977) “ Dermal and Eye Toxicity Tests” In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC p. 48 to 49. Any other ocular effects were also noted at this point along with any clinical signs of toxicity if present. Individual bodyweights were recorded on day 0 and 3. The numerical values corresponding to each animal, tissue and observation time were recorded. These scores were then assessed using a modified version of the system described by Kay JH and Calandra JC (1962), J. Soc. Cosmet. Chem. 13, 281-289. If evidence of irreversible ocular damage is noted, the test material will be classified as corrosive to the eye.

TOOL USED TO ASSESS SCORE: standard ophthalmoscope
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: 24, 48, 72 hours
Score:
0
Max. score:
80
Reversibility:
other: not applicable
Remarks on result:
other: No corneal effects observed
Irritation parameter:
iris score
Basis:
mean
Time point:
other: 24, 48, 72 hours
Score:
0
Max. score:
10
Reversibility:
other: not applicable
Remarks on result:
other: No iridial effects observed
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
other: 24, 48, 72 hours
Score:
4
Max. score:
20
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: Not applicable
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: Not applicable
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: Not applicable
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible
Irritant / corrosive response data:
The test material produced a maximum group mean score of 10.0

To calculate the maximum group mean score, encompassing all reactions, the following equations were used:
Cornea = (E x F) x 5
Where:
E = Degree of opacity
F = Area of Cornea Involved

Iris = (D x 5)

Conjunctivae = (A + B + C) x 2
Where:
A = Redness
B = Chemosis
C = Discharge

Measurement of pH

The pH of the test material was determined prior to commencement of the study and found to be as follows:

 

Preparation

pH Measurement

immediately

after 10 minutes

after 20 minutes

 10 % w/w aqueous preparation of the test material

8.9

9.6

9.6

 

 

Table 1: Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex

68648 Male

68668 Male

68669 Male

IPR = 2

IPR = 2

IPR = 2

Time After Treatment

1 Hr

24 Hrs

48 Hrs

72 Hrs

1 Hr

24 Hrs

48 Hrs

72 Hrs

1 Hr

24 Hrs

48 Hrs

72 Hrs

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

1

0

0

2

1

0

0

2

1

0

0

B = Chemosis

2

1

0

0

2

1

0

0

2

1

0

0

C = Discharge

1

0

0

0

1

0

0

0

1

0

0

0

Score (A + B + C) x 2

10

4

0

0

10

4

0

0

10

4

0

0

Total Score

10

4

0

0

10

4

0

0

10

4

0

0

IPR = Initial pain reaction

 

Table 2: Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbit Number and Sex

Individual Total Scores At:

1 Hour

24 Hours

48 Hours

72 Hours

68648 Male

10

4

0

0

68668 Male

10

4

0

0

68669 Male

10

4

0

0

Group Total

30

12

0

0

Group Mean Score

10.0

4.0

0.0

0.0

 

Table 3:Individual Bodyweights and Bodyweight Changes

Rabbit Number and Sex

Individual Bodyweight (kg)

Bodyweight Change (kg)

Day 0

Day 3

68648 Male

2.33

2.37

0.04

68668 Male

2.39

2.42

0.03

68669 Male

2.18

2.30

0.12

Interpretation of results:
not classified
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Slight conjunctival redness and chemosis (score 1) was observed 24 hours following administration of the test material in the treated eyes of all three animals; all treated eyes appeared normal 48 hours after administration. Under the conditions of the study the test material was determined to be not irritating to eyes.
Executive summary:

The eye irritation potential of the test material was investigated in vivo in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 405 and EU Method B.5.

No corneal or iridial effects was noted during the study. Moderate conjuctival irritation was noted in all treated eyes one hour after treatment with minimal conjunctival irritation noted at the 24-hour observation. Under the conditions of the study the test material was determined to be not irritating to eyes.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 August 2009 to 27 August 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study performed according to GLP and follows an appropriate protocol for in vitro eye irritation potential without any deviations from the study design. As the method is not yet included in a standardised guideline the data have been assigned a reliability score of 2.
Qualifier:
no guideline available
Principles of method if other than guideline:
The aim of the study was to determine the eye irritation potential of the test material using the SkinEthic Reconstituted Human Corneal model (HCE SkinEthic Laboratories, Nice, France) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.
GLP compliance:
yes (incl. QA statement)
Species:
other: Reconstituted Corneal Epithelium
Details on test animals or tissues and environmental conditions:
TEST CULTURE
- Source: SkinEthic Laboratories, Nice, France
- Date Received : 25/08/09
- Culture details: Day 6 cultures
- Storage of culture: Cultures were stored at room temperature on arrival and then transferred into 24-well plates containing 300 µL of maintenance medium. No air bubbles were present under the tissue inserts. Tissues were incubated overnight at 37 °C, 5% CO2 in air.
Tissue preparation: Using sterile techniques, 1 mL of maintenance medium at room temperature dispensed into the required number of wells in a 6-well plate. Each well was labelled with the details of treatment and the appropriate exposure time. Separate treatment plates were used for the test material and controls (negative and positive) to avoid cross contamination. Before treatment, 7 day old cultures were transferred into the treatment plates containing maintenance medium.
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied : Tissues were treated with 30 mg of the test material

VEHICLE
Test material was used as supplied

CONTROLS:
- Amount(s) applied: 30 µL of Solution A was applied as a negative control and 30 µL of of SDS 1.0% (w/v) as a positive control. Solution A was comprised of Na2HPO4 0.142 g/L, Glucose 1.802 g/L, HEPES 7.149 g/L, KCl 0.224 g/L and NaCl 7.597 g/L
Sodium Dodecyl Sulphate (SDS) was prepared as a 1% w/v solution in sterile distilled water
Duration of treatment / exposure:
Cultures were exposed for 10 minutes to the test material
Observation period (in vivo):
Skin cultures were examined after three hours
Number of animals or in vitro replicates:
All test substances were tested in triplicate (including controls)
Details on study design:
REMOVAL OF TEST MATERIAL
- Washing (if done): Cultures were rinsed by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated "holding plate" containing 300 µL of maintenance medium (at room temperature) until all tissues were rinsed.
- Time after start of exposure: 10 minutes

STAINING PROCEDURE:
Staining: After rinsing, the tissues (two per group) were transferred into a pre-labelled 24-well plate containing 300 µL of a 0.5 mg/mL MMT solution prepared in maintenance medium. The MMT loading plate was placed into an incubator for approximately three hours at 37 °C, 5 % CO2 in air.

SCORING SYSTEM:
- Tissue viability (OD): After incubation with MMT, the tissues were visually examined and the degree of MMT staining was evaluated (qualitative evaluation of tissue viability). The inserts were blotted on absorbent paper to remove residual MMT and transferred to a pre-labelled 24-well plate containing 0.75 mL of isopropanol in each of a sufficient number of wells. An extra 0.75 mL of isopropanol was added onto each tissue and the plate sealed to prevent isopropanol evaporation. The plate was wrapped in aluminium foil and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue.

- Histology: If deemed necessary, the histopathology of the remaining insert was examined. At the end of the extraction period, each tissue insert was pierced with a pipette fitted with a 1000 µL tip and the extraction solution forced vigorously up and down through the tissue insert until a homogeneous solution was obtained. The empty inserts were discarded for each tissue triplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µL of Isopropanol alone was added to three wells designated as blanks. The optical density was measured (quantitative measurement of tissue viability) at 540 nm (OD540) using Anthos 2001 microplate reader. One tissue for each treatment group was retained for assessment of tissue histopathology. Tissues were cut out of the polycarbonate inserts with a sharp scalpel. The tissues were cut in half. Both halves were placed into a pre-labelled 1.5 mL Eppendorf tube containing 1 mL of 10% Formalin and stored at room temperature.
To determine histopathological changes the tissues are observed for any changes in thickness or organisation of the cells. The negative control tissues should have a constant thickness devoid of terminally differentiated cells, and feature a regular and compact shape. Cells must maintain attachment via multiple desmosomes. Positive control tissues should have a disintegration of most of the upper cell layers of the epithelial tissue. The remaining basal cells should be loosely attached to the polycarbonate substratum.

TOOL USED TO ASSESS SCORE: Anthos 2001 microplate reader.
Irritation parameter:
other: Mean tissue viability
Run / experiment:
Mean
Value:
> 0.99 - < 1.1
Other effects / acceptance of results:
-Interpretation of data: Quantitative MMT Assessment (percentage of viable tissue)
The relative mean tissue viabilities are compared to the mean of the untreated negative control tissues (n = 2). The relative mean viabilities are calculated using the following: (mean OD540 of test material/mean OD540 of negative control) x 100

The relative mean viability of the test material treated tissues after a 10 minute exposure was 97.6%.

The test material was found to not directly reduce MMT. It was deemed unnecessary to examine tissue histopathology.

Table 1: Assessment of Eye Irritation Potential – Viability of RHC Tissues

Material

Mean Tissue Viability

Mean OD540

Viability (%)

Negative control

1.042

1.010

100*

0.977

Positive control

0.282

0.218

21.6

0.154

Test material

0.992

0.986

97.6

1.060

* The mean viability of the negative control tissues is set at 100 %

 

Table 2: Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material

Score

Tissue 1

Tissue 2

Negative control

-

-

Positive control

+

+

Test material

-

-

- Blue tissue (viable)

+ Blue/White tissue (semi viable)

++ Tissue completely white (dead)

Interpretation of results:
not irritating
Remarks:
Criteria used for interpretation of results: other: Criteria set out in the protocol of this study.
Conclusions:
According to the protocol followed, the test material was found to be a non-irritant (NI) to the reconstituted human corneal epithelial model, SkinEthic.
Executive summary:

The aim of the study was to determine the eye irritation potential of the test material using the SkinEthic Reconstituted Human Corneal model (HCE SkinEthic Laboratories, Nice, France) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.

According to the protocol followed, the test material was found to be a non-irritant (NI) to the reconstituted human corneal epithelial model, SkinEthic.

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

Respiratory irritation

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

Additional information

Skin

In vitro testing concluded that the test material was unlikely to be irritating to skin following exposure. An in vivo test was subsequently performed to confirm findings of the in vitro study.

The skin irritation potential of the test material was investigated in vivo in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 404 and EU Method B.4.

During the study no signs of skin irritation were noted at any observation point. The test material was therefore concluded to be a non-irritant to the skin.

Eye

In vitro testing concluded that the test material was unlikely to be an eye irritant. An in vivo test was subsequently performed to confirm findings of the in vitro study.

The eye irritation potential of the test material was investigated in vivo in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 405 and EU Method B.5.

No corneal or iridial effects were noted during the study. Moderate conjuctival irritation was noted in all treated eyes one hour after treatment with minimal conjunctival irritation noted at the 24-hour observation. The test material was therefore concluded to be not irritating to eyes.


Justification for selection of skin irritation / corrosion endpoint:
The key study was selected as such because it represents the only available in vivo study.

Justification for selection of eye irritation endpoint:
The key study was selected as such because it represents the only available in vivo study.

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 either skin or eye irritation.