Registration Dossier

Administrative data

Description of key information

The skin irritation potential of the test item using the EPISKINTM. The relative mean viability of the test item treated tissues was 105.1% after the 15-Minute exposure period. The test item was considered to be non-irritant to skin. Based on the available in vitro and in vivo eye corrosion/irritation studies the substance is not classified as irritating to skin.

The test item was considered not to be an ocular corrosive or severe irritant in the in vitro BCOP assay. In an in vivo assay in rabbits the test item produced a maximum group mean score of 4.0 and was classified as a minimal irritant. Based on the available in vitro and in vivo eye corrosion/irritation studies the substance is not classified as irritating to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion
Remarks:
other: in vitro in accordance with OECD 439
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 September 2011 - 26 September 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols and under GLP conditions.
Qualifier:
according to
Guideline:
other: EU Guideline Testing of Chemicals B46
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD 439
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: reconstructed human epidermis model
Strain:
other: reconstructed human epidermis model
Details on test animals and environmental conditions:
Not applicable
Type of coverage:
other: Topical
Preparation of test site:
other: Not applicable
Vehicle:
other: Not applicable
Controls:
no
Amount / concentration applied:
TEST ITEM

The test item was applied neat.

Amount(s) applied (volume or weight with unit):
Approximately 10 µl of the test item was applied to the epidermis surface.

Concentration (if solution):
The test item was used as supplied.

VEHICLE
No vehicle used
Duration of treatment / exposure:
15 minutes & 42 hour post exposure incubation
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
TEST SITE
Area of exposure:
Approximately 10 µl of the test item was applied to the epidermis surface.

% coverage:
The test item was applied topically to the corresponding tissues ensuring uniform covering.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing PBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test item.

Time after start of exposure:
15 Minutes post exposure

SCORING SYSTEM
Quantitative MTT Assessment (percentage tissue viability):
For the test item the relative mean tissue viabilities obtained after the 15 minute treatment followed by the 42 hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:

mean OD540 of test item / mean OD540 of negative control x 100 = Relative mean tissue viability (percentage of negative control)

Classification of irritation potential is based upon relative tissue viability following the 15 minute exposure period followed by the 42 hour post-exposure incubation period according to the following:

Mean tissue viability is ≤50% : Irritant

Mean tissue viability is >50% : Non-Irritant
Irritation parameter:
other: Viability of cells
Basis:
mean
Remarks:
Viability of cells
Time point:
other: Day 6
Reversibility:
other: Not applicable
Remarks on result:
other: See relative mean viability below.
Irritant / corrosive response data:
The relative mean viability of the test item treated tissues was 105.1% after a 15 Minute exposure period.
Other effects:
No

RESULTS

Direct MTT Reduction

The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Test Item, Positive Control Item and Negative Control Item

The individual and mean OD540 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Table 1.

The relative mean viability of the test item treated tissues was 105.1% after a 15-Minute exposure period.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 12.4% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 7.0%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 0.678 and the standard deviation value of the percentage viability was 5.7%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 5.5%. The test item acceptance criterion was therefore satisfied.

Table1 : Mean OD540 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

OD540 of tissues

Mean OD540 of triplicate tissues

±SD of OD540

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.718

0.678

0.039

105.9

100*

5.7

0.641

94.5

0.675

99.6

Positive Control Item

0.056

0.084

0.048

8.3

12.4

7.0

0.057

8.4

0.139

20.5

Test Item

0.745

0.712

0.037

109.9

105.1

5.5

0.672

99.1

0.720

106.2


SD=   Standard deviation

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

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be non-irritant to skin.
Executive summary:

Introduction:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 -[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 -Hour post-exposure incubation period is also determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result. This method was designed to be compatible with the following:

  • OECD Guidelines for the Testing of Chemicals No. 439 “In Vitro Skin Irritation” (adopted 22 July 2010)
  • Method B.46 of Commission Regulation (EC) No. 440/2008/EC

Method: Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. 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.

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

Results: The relative mean viability of the test item treated tissues was 105.1% after the 15-Minute exposure period.

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion: The test item was considered to be Non-Irritant.

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:
09 January 2012 and 19 January 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is performed according to OECD guidelines and GLP.
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Harlan Laboratories UK Ltd., Leicestershire, UK. At the start of the study the animals weighed 2.11 or 2.82 kg and were twelve to twenty weeks old. After an acclimatisation period of at least five days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930 Teklad Global Certified Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 17 to 23°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Vehicle:
unchanged (no vehicle)
Controls:
other: The left eye remained untreated and was used for control purposes.
Amount / concentration applied:
A volume of 0.1 ml of the test item was placed into the conjunctival sac of the right eye
Duration of treatment / exposure:
1 hour
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
2
Details on study design:
Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.
Initially, a single rabbit was treated. A volume of 0.1 ml of the test item was placed 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 the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale shown in Appendix 1.
After consideration of the ocular responses produced in the first treated animal, a second animal was treated.
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation given in Appendix 2, (from Draize J H (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. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Any clinical signs of toxicity, if present, were also recorded.
Individual bodyweights were recorded on Day 0 (the day of dosing) and at the end of the observation period.
Irritation parameter:
cornea opacity score
Basis:
animal: 71538 Male
Time point:
other: Score at 1, 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: No effects observed
Irritation parameter:
cornea opacity score
Basis:
animal: 71584 Male
Time point:
other: Score at 1, 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: No effects observed
Irritation parameter:
iris score
Basis:
animal: 71538 Male
Time point:
other: Score at 1, 24, 48 and 72 hours
Score:
0
Max. score:
2
Reversibility:
other: No effects observed
Irritation parameter:
iris score
Basis:
animal: 71584 Male
Time point:
other: Score at 1, 24, 48 and 72 hours
Score:
0
Max. score:
2
Reversibility:
other: No effects observed
Irritation parameter:
other: redness
Basis:
animal: 71538 Male
Time point:
other: Score at 1, 24, 48 and 72 hours
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
other: redness
Basis:
animal: 71584 Male
Time point:
other: Score at 1, 24, 48 and 72 hours
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal: 71538 Male
Time point:
other: Score at 1, 24, 48 and 72 hours
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 24 hours
Irritation parameter:
chemosis score
Basis:
animal: 71584 Male
Time point:
other: Score at 1, 24, 48 and 72 hours
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritant / corrosive response data:
Individual and group mean scores for ocular irritation are given in Table 1 and Table 2.
No corneal or iridial effects were noted during the study.
Minimal conjunctival irritation was noted in both treated eyes one and 24 hours after treatment.
Both treated eyes appeared normal at the 48-Hour observation.
Other effects:
Individual bodyweights and bodyweight changes are given in Table 3.
Both animals showed expected gain in bodyweight during the study.

Interpetation of Results

The numerical values corresponding to each animal, tissue and observation time were recorded. The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of cornea involved). For each tissue the score was calculated as follows:

Score for conjunctivae       =         (A + B + C) x 2
Score for iris                         =         D x 5
Score for cornea                 =         (E x F) x 5

Using the numerical data obtained a modified version of the system described by Kay J H and Calandra J C (1962), J. Soc. Cosmet. Chem.13, 281‑289 (see Appendix 3) was used to classify the ocular irritancy potential of the test item. This was achieved by adding together the scores for the cornea, iris and conjunctivae for each time point for each rabbit. The group means of the total scores for each observation were calculated. The highest of these group means (the maximum group mean score) together with the persistence of the reactions enabled classification of the eye irritancy potential of the test item.

If evidence of irreversible ocular damage is noted, the test item will be classified as corrosive to the eye.

The results were also interpreted according to Regulation (EC) No. 1272/2008 on the classification, labelling and packaging of substances and mixtures.

Table 1              Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex

71538Male

71584Male

IPR= 1

IPR = 1

Time After Treatment

1
Hour

24
Hours

48
Hours

72
Hours

1
Hour

24
Hours

48
Hours

72
Hours

CORNEA

 

 

 

 

 

 

 

 

E = Degree of Opacity

0

0

0

0

0

0

0

0

F = Area of Cornea Involved

0

0

0

0

0

0

0

0

Score (E x F) x 5

0

0

0

0

0

0

0

0

IRIS

 

 

 

 

 

 

 

 

D

0

0

0

0

0

0

0

0

Score (D x 5)

0

0

0

0

0

0

0

0

CONJUNCTIVAE

 

 

 

 

 

 

 

 

A = Redness

1

1

0

0

1

1

0

0

B = Chemosis

1

0

0

0

1

0

0

0

C = Discharge

0

0

0

0

0

0

0

0

Score (A + B + C) x 2

4

2

0

0

4

2

0

0

Total Score

4

2

0

0

4

2

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

71538Male

4

2

0

0

71584Male

4

2

0

0

Group Total

8

4

0

0

Group Mean Score

4.0

2.0

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

71538Male

2.82

2.85

0.03

71584Male

2.11

2.20

0.09

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item produced a maximum group mean score of 4.0 and was classified as a minimal irritant (Class 3 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.
The test item did not meet the criteria for classification according to Regulation (EC) No. 1272/2008 on the classification, labelling and packaging of substances and mixtures.
Executive summary:

Introduction. The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit. The method was designed to be compatible with the following:

OECD Guidelines for the Testing of Chemicals No.405 "Acute Eye Irritation/Corrosion" (adopted 24 April 2002)

Method B5 Acute Toxicity (Eye Irritation) of Commission Regulation (EC) No.440/2008

Result. A single application of the test item to the non-irrigated eye of two rabbits produced minimal conjunctival irritation. Both treated eyes appeared normal at the 48‑Hour observation.

Conclusion. The test item produced a maximum group mean score of 4.0 and was classified as a minimal irritant (Class 3 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item did not meet the criteria for classification according to Regulation (EC) No. 1272/2008 on the classification, labelling and packaging of substances and mixtures.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The registered substance is not irritating to skin and eyes.

Justification for selection of skin irritation / corrosion endpoint:

K1: Study conducted in compliance with agreed protocols and under GLP conditions.

Justification for selection of eye irritation endpoint:

K1: Study conducted in compliance with agreed protocols and under GLP conditions.

Justification for classification or non-classification

Based on the available results from in vitro skin and eye irritation studies and an in vivo eye irritation study in rabbits, the data is conclusive but not sufficient for classification for these endpoints.