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Description of key information

This in vitro study was performed to assess the skin irritation potential of the test item by means of the Human Skin Model Test.


Three tissues of the human skin model EpiSkin™ were treated each with the test item, the negative or the positive control for 15 minutes.


Approximately 10 mg of the neat test item were applied to each tissue, spread to match the tissue size. Additionally, the test item tissues were wetted with 20 µL of deionised water.


After treatment with the test item the relative absorbance values did not decrease relevantly (92.3%; threshold for irritancy:≤50%). Therefore, the test item is not considered to possess an irritant potential.


In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin.


In conclusion, it can be stated that in this study and under the experimental conditions, the test item is not irritant to skin.


 


Eye irritation


The primary eye irritation potential of the test material was investigated according to a method compatible with OECD test guideline no. 405.


Conclusion


Based upon the EU-classification criteria, the test item does not have to be classified with respect to eye irritation.

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:
From 28 MAR 2012 to 02 APR 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 439) and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
other: OECD Guideline for the Testing of Chemicals 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (Original Guideline adopted July 22, 2010)
GLP compliance:
yes
Amount / concentration applied:
10 mg of the undiluted test item were applied to three EPISKIN (Skinethic) tissues, test item tissues were wetted with 20 µL of deionised water
Duration of treatment / exposure:
The skin equivalents were exposed to the test item for 15 minutes. After completion of the treatment the test item was rinsed off and the skin equivalents were incubated for further 42 hours.
Details on study design:
10 µL of the undiluted test item, the positive control (5% sodium lauryl sulfate) and the negative control (deionised water) were applied to three EPISKIN (Skinethic) tissues. The test item as well as the controls were rinsed off after 15 minutes treatment. After further 42 hours incubation the tissues were treated with the MTT solution for 3 hours following approximately 69 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at a wavelength of 570 nm.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
all
Value:
>= 92.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
After treatment with the test item the relative absorbance values did not decrease relevantly (92.3%; threshold for irritancy: ≤ 50%). Therefore, the test item is not considered to possess an irritant potential.

Results after treatment with test substance and controls

 

Dose group

Treatment Interval

Absorbance 570 nm
Tissue 1*

Absorbance 570 nm
Tissue 2*

Absorbance 570 nm
Tissue 3*

Mean Absorbance of 3 Tissues

Relative Absorbance [%] Tissue 1, 2 + 3**

Standard Deviation [%]

Rel. Absorbance

[% of Negative Control]***

Negative Control

15 min

1.095

1.046

1.148

1.096

99.9

95.4

104.7

4.7

100.0

Positive Control

15 min

0.441

0.383

0.340

0.388

40.2

34.9

31.0

4.6

35.4

Test Item

15 min

0.935

1.024

1.077

1.012

85.2

93.4

98.2

6.5

92.3

*       Mean of two replicate wells after blank correction

**      relative absorbance per tissue [rounded values]
***    relative absorbance per treatment group [rounded values]

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

The relative absorbance value of the test item, corresponding to the cell viability, did not decrease (threshold for irritancy:≤50%), consequently the test item was non irritant to skin.

Interpretation of results:
not irritating
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin.
Executive summary:

This in vitro study was performed to assess the skin irritation potential of the test item by means of the Human Skin Model Test.


Three tissues of the human skin model EpiSkin™ were treated each with the test item, the negative or the positive control for 15 minutes.


Approximately 10 mg of the neat test item were applied to each tissue, spread to match the tissue size. Additionally, the test item tissues were wetted with 20 µL of deionised water.


The test item and the positive and negative controls were washed off the skin tissues after 15 minutes treatment. After further incubation for about 42.5 hours the tissues were treated with the MTT solution for 3 hours following 69 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.


10 µL of either the negative control (deionised water) or the positive control (5% Sodium lauryl sulfate) were applied to each tissue.


After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD greater or equal 0.6 till less or equal 1.5 for the15 minutes treatment interval thus showing the quality of the tissues.


Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 35.4% thus ensuring the validity of the test system.


The standard deviations between the % variabilities of the test item, the positive and negative controls were below 7% (threshold of the "OECD TG 439 Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%), thus ensuring the validity of the study.


After treatment with the test item the relative absorbance values did not decrease relevantly (92.3%; threshold for irritancy:≤50%). Therefore, the test item is not considered to possess an irritant potential.


In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant 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 vitro / ex vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro eye irritation study does not need to be conducted because adequate data from an in vivo eye irritation study are available
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 06 JUN 2012 to 15 JUN 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 405) and according to GLP.
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
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Three 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 2.31 to 2.75 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, which was found to weigh approximately 35 mg
Duration of treatment / exposure:
single application / no washing
Observation period (in vivo):
Examinations were done approximately 1 hour and 24, 48 and 72 hours following treatment
Number of animals or in vitro replicates:
3 animals were tested in total. (After consideration of the ocular responses produced in the first treated animal, two additional animals were treated. )
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, which was found to weigh approximately 35 mg (as measured by gently compacting the required volume into an adapted syringe) 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.
After consideration of the ocular responses produced in the first treated animal, two additional animals were treated. In order to minimise pain on application of the test item, one drop of local anaesthetic (Tetracaine hydrochloride 0.5%, Bausch & Lomb, Surrey, UK) was instilled into both eyes of these animals 1 to 2 minutes before treatment. Due to a technician error the initial pain reaction was not recorded for the third animal. This deviation was considered not to affect the purpose or integrity of the study.
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:
mean
Time point:
other: Mean score at 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: No effects observed
Irritation parameter:
iris score
Basis:
mean
Time point:
other: Mean score at 24, 48 and 72 hours
Score:
0
Max. score:
2
Reversibility:
other: No effects observed
Irritation parameter:
other: redness
Basis:
animal: 72094 Male
Time point:
other: Mean score at 24, 48 and 72 hours
Score:
0.67
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
other: redness
Basis:
animal: 72123 Male
Time point:
other: Mean score at 24, 48 and 72 hours
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
other: redness
Basis:
animal: 72125 Male
Time point:
other: Mean score at 24, 48 and 72 hours
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
animal: 72094 and 72123 Male
Time point:
other: Mean score at 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: No effects observed
Irritation parameter:
chemosis score
Basis:
animal: 72123 Male
Time point:
other: Mean score at 24, 48 and 72 hours
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritant / corrosive response data:
Ocular Reactions
Individual and mean scores for eye irritation are given in Table 1 and Table 2.
Orange coloured staining of the fur was noted around all treated eyes throughout the study.
No corneal effects were noted during the study.
Iridial inflammation was noted in one treated eye one hour after treatment.
Moderate conjunctival irritation was noted in all treated eyes one hour after treatment. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in two treated eyes at the 24 Hour observation. Minimal conjunctival irritation was noted in two treated eyes at the 48 Hour observation.
One treated eye appeared normal at the 48 Hour observation and two treated eyes appeared normal at the 72 Hour observation.
All signs of irritation were fully reversible within 72 Hours.
No corrosive effects were noted during the study.
Other effects:
Clinical Observations
No clinical signs of toxicity were noted during the study.

Bodyweight
Individual bodyweights and bodyweight changes are given in Table 2.
All animals showed expected gains in bodyweight during the study.

Table 2              Individual Bodyweights and Bodyweight Changes

Rabbit Number
and Sex

Individual Bodyweight (kg)

Bodyweight Change (kg)

Day 0

Day 3

72094Male

2.75

2.81

0.06

72123 Male

2.41

2.47

0.06

72125 Male

2.31

2.35

0.04
Interpretation of results:
not irritating
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
According to the classification criteria of Regulation (EC) No 1272/2008 the test material is not irritating to eyes under these test conditions.
Executive summary:

Introduction


The primary eye irritation potential of the test material was investigated according to a method compatible with OECD test guideline no. 405 and Method B5.


Method


The test item was applied by instillation of 0.1 ml into the right eye of each of three young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours after test item instillation. 


The mean score was calculated separately for each animal across three scoring times (24, 48 and 72 hours after instillation) for corneal opacity, iritis, redness and chemosis of the conjunctivae.


Results


The individual mean scores for corneal opacity and iritis were 0.00 for all three animals. The individual mean scores for the conjunctivae were 0.67, 0.33 and 1.00 for reddening and 0.00, 0.00 and 0.33 for chemosis.


The instillation of test substance into the eye resulted in minimal to moderate irritation, early‑onset and transient ocular changes, such as iridial inflammation and reddening of the conjunctivae, ocular discharge and chemosis. These effects were reversible and were no longer evident 72 hours after treatment, the end of the observation period for all animals. No abnormal findings were observed in the cornea of any animals at any of the examinations. No corrosion was observed at any of the measuring intervals. No clinical signs of toxicity were observed.


Thus, the test item did not induce significant or irreversible damage to the rabbit eye.


Conclusion


Based upon the referred classification criteria (Regulation (EC) No. 1272/2008 of the European Parliament and of the Council of 16 December 2008), the test item does not have to be classified with respect to eye irritation.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

No classification


The test item did not caus irritant effects on skin and eyes.