Registration Dossier

Administrative data

Description of key information

Skin: Not classified as corrosive, EpiSkin, OECD 431, Hargitai 2014
Skin: Not classified as irritant, EpiSkin, OECD 439, Hargitai 2014
Eye: Not classified as a severe irritant and not classified as non-irritant (inconclusive), ICE, OECD 438, Váliczkó 2014
Eye: Not classified as irritant, rabbit, OECD 405, Matting 2015

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:
2013-11-20 until 2013-11-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, no restrictions, fully adequate for assessment.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: COMMISSION REGULATION (EU) No 640/2012 B.46. (2012)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
human
Details on test animals and environmental conditions:
HUMAN SKIN: EPISKIN® (SM) (Manufacturer: SkinEthic, France, Batch No.: 13-EKIN-041, Expiry Date: 25 November 2013) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
Controls:
other: Not applicable
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg

Duration of treatment / exposure:
15 mins
Details on study design:
Disks of EPISKIN (3 units/chemical) were treated with the test item and incubated for 15 mins at room temperature. Exposure of test material was terminated by rinsing with phosphate buffered saline (PBS). Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of the epidermis on 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.

SDS 5% and PBS treated epidermis were used as positive and negative controls, respectively. An additional disk was used to provide an estimate of colour contribution from the test item. For each treated tissue viability was expressed as a % relative to negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test substance is considered to be irritant to skin.
Irritation / corrosion parameter:
other: other: Mean cell viability
Value:
138
Remarks on result:
other:
Remarks:
Basis: mean. Remarks: non irritant. (migrated information)
Irritant / corrosive response data:
Based on the OD value the viability was 138±11.50% for the test item treated skin.
The mean OD value of the three negative control tissues was 0.800. The positive control result showed a mean of 14% viability. Each standard deviation value (SD) of the % viability was below 18. Control OD values were below historically established boundaries. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

As the test item was coloured, one additional test item-treated tissue was used for the non specific OD evaluation. Optical density (measured at 540 nm) of this tissue was determined as 0.016, Non Specific Colour % was calculated as 2.0%. Therefore additional data calculation was not necessary.
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.

Table 1: Optical Density (OD) and the calculated % viability of the cells

Substance

Optical density

Viability (%)

Negative control (PBS)

1

0.819

102

2

0.837

105

3

0.743

93

mean ± SD

0.800

100±6.24

Positive control (5% SDS)

1

0.114

14

2

0.111

14

3

0.104

13

mean ± SD

0.110

14±0.58

Test item

1

1.101

138

2

1.197

150

3

1.012

127

mean ± SD

1.103

138±11.50

 

Interpretation of results:
study cannot be used for classification
Remarks:
Migrated information
Conclusions:
In this in vitro EPISKIN®(SM) model test, the results indicate that the test item is Non Irritant (No Category).
Executive summary:

The reconstructed human epidermis model EPISKIN® (SM) is designed to predict and classify the skin irritant potential of chemicals, by measuring its cytotoxic effect, as reflected in the MTT assay, on the EPISKIN reconstituted human epidermis. This method is approved by international regulatory agencies as a replacement for the identification of irritants / corrosives in the in vivo Rabbit skin assay (OECD 404).

Disks of EPISKIN (3 units / chemical) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with phosphate buffered saline (PBS). Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of the epidermis on 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. SDS 5% and PBS treated epidermis were used as positive and negative controls, respectively. An additional disk was used to provide an estimate of colour contribution from the test item. For each treated tissue viability was expressed as a % relative to negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test substance is considered to be irritant to skin.

Following exposure with the test item, the mean cell viability was 138% compared to the negative control and, therefore, non-irritant. All validity criteria were within acceptable limits and therefore the study can be considered as valid. In this in vitro EPISKIN®(SM) model test, the results indicate that the test item is Non Irritant (No Category).

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:
2014-03-11 until 2014-03-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, no restrictions, fully adequate for assessment.
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
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:
TEST ANIMALS
- Source: S&K-LAP Kft., 2173 Kartal, Császár út 135, Hungary
- Age at study initiation: 11-12 weeks
- Weight at study initiation: 2992-3077 g
- Housing: Individually housed in AAALAC approved metal wire rabbit cages
- Diet: UNI diet for rabbits ad libitum. Source of feed: Agribrands Europe Hungary PLC, H-5300 Karcag, Madarasi út, Hungary
- Water: Tap water ad libitum
- Acclimation period: At least 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.0–20.4
- Humidity (%): 24-48
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 11 March 2014 To: 18 March 2014
Vehicle:
unchanged (no vehicle)
Controls:
other: The untreated contralateral eye served as the control.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g
Duration of treatment / exposure:
Single instillation (eye lids gently held together for at least one second in order to prevent loss of the material).
Observation period (in vivo):
72 hours (scored approximately 1, 24, 48, 72 hours after installation).
Number of animals or in vitro replicates:
3
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, rinsed with physiological saline.
- Time after start of exposure: 1 hour after instillation

SCORING SYSTEM: The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (02 October 2012)

TOOL USED TO ASSESS SCORE: hand-slit lamp / fluorescein
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
other: mean of 24, 48, 72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
other: mean of 24, 48, 72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
other: mean of 24, 48, 72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #1
Time point:
other: mean of 24, 48, 72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #2
Time point:
other: mean of 24, 48, 72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #3
Time point:
other: mean of 24, 48, 72 h
Score:
0
Max. score:
2
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: mean of 24, 48, 72 h
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: mean of 24, 48, 72 h
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
other: mean of 24, 48, 72 h
Score:
0
Max. score:
4
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Time point:
other: mean of 24, 48, 72 h
Score:
0.7
Max. score:
3
Reversibility:
fully reversible within: 72 h
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Time point:
other: mean of 24, 48, 72 h
Score:
0.3
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #3
Time point:
other: mean of 24, 48, 72 h
Score:
0.7
Max. score:
3
Reversibility:
fully reversible within: 72 h
Irritant / corrosive response data:
No Initial Pain Reaction/Pain reaction (IPR/PR) was observed.

1 hour after treatment: Conjunctival redness (score 1 or 2), chemosis (score 1 or 2) and discharge (score 3) were seen in all rabbits. Conjunctival redness (score 1) and discharge (score 1) were seen in all rabbits at 24 hours after treatment. Conjunctival redness (score 1) was seen in two rabbits and discharge (score 1) was seen in one rabbit at 48 hours after treatment. All symptoms had fully reversed in all animals by the 72 hours observations.

Fluorescein staining was negative in all animals at 24, 48 and 72 hours after instillation.
.
The control eyes were symptom-free during the study.
Other effects:
No mortality, no clinical signs of systemic toxicity and bodyweights of all rabbits were within the normal range of variability.

Table 1: Eye irritation scores according to the Draize scheme

Time

Cornea

Iris

Conjunctiva

 

 

 

 

Redness

Chemosis

 

Animal number

832

849

864

832

849

864

832

849

864

832

849

864

after 1 hour

0d

0

0

0

0

0

1

2

2

1

2

1

after 24 hours

0d

0d

0d

0

0

0

1

1

1

0

0

0

after 48 hours

0

0d

0d

0

0

0

1

0

1

0

0

0

after 72 hours

0

0d

0

0

0

0

0

0

0

0

0

0

mean scores 24-72 hours

0

0

0

0

0

0

0.7

0.3

0.7

0

0

0

d conjunctival discharge

 

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was graded as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to the modified Kay and Calandra classification system.
Executive summary:

The primary eye irritation effect of the test item was evaluated according to OECD test guideline no.: 405 (2012), EPA OPPTS 870.2400 (1998), Directive 2004/73/EC B.5 (L 152 2004) and EC No 440/2008, B.5 (2008). The test item was administered as an installation of a single dose of 0.1 g into the conjunctival sac of the left eye of 3 adult male New Zealand White rabbits. The untreated right eye served as the control. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours after test material installation in all animals. Observations with fluorescein staining were made approximately 24 hours before treatment and then 24, 48 and 72 hours after the treatment in all animals. Rabbits were treated with analgesic and anaesthetic as per the regulatory guideline. Results obtained from these three animals were used to classify the test item for irritation potential.

No Initial Pain Reaction/Pain reaction (IPR/PR) was observed. Conjunctival redness (score 1 or 2), chemosis (score 1 or 2) and discharge (score 3) were seen in all rabbits at 1 hour after treatment. Conjunctival redness (score 1) and discharge (score 1) were seen in all rabbits at 24 hours after treatment. Conjunctival redness (score 1) was seen in two rabbits and discharge (score 1) was seen in one rabbit at 48 hours after treatment. All symptoms had fully reversed in all animals by the 72 hours observations. Fluorescein staining was negative in all animals at 24, 48 and 72 hours after instillation. The control eye of animals was symptom-free during the study. No mortality occurred during the study. The bodyweights of all rabbits were considered to be within the normal range of variability.

The test item was graded as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to the modified Kay and Calandra classification system.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation / Corrosion

Two studies are available, both done according to GLP, using guideline methods which are validated for regulatory use:

In order to assess the in vitro skin corrosion potential of the substance, EpiSkin disks (Hargitai 2014) were treated with the test item, negative controls (0.9 % sodium chloride) and positive controls (glacial acetic acid) and incubated for four hours at room temperature (two replicates each). The mean cell viability was 115 % compared to the negative control (100 %) and the positive control (0.7 %). All validity criteria were within acceptable limits and therefore the study can be considered as valid. The substance is not corrosive to the in vitro skin model EpiSkin.

In order to assess the in vitro skin irritation potential of the substance, EpiSkin disks (Hargitai 2014) were treated with the test item, negative controls (PBS) and positive controls (5% SDS) for 15 min (three replicates each). The mean cell viability was 138 % compared to the negative control (100 %) and the positive control (14 %). All validity criteria were within acceptable limits and therefore the study can be considered as valid. The substance isnot irritant to the in vitro skin model EpiSkin.

Based on the available in vitro data which is considered to be adequate, reliable and conclusive for skin irritation/corrosion, the substance is considered to not be an irritant.

Eye Irritation

In vitro data

In order to assess the in vitro eye irritation potential of the substance, OECD Guideline 438 Isolated Chicken Eye Test Method was carried out by Váliczkó (2014). Simultaneously were performed three tests with the substance , three positive controls (imidazole) and one negative control (0.9 % sodium chloride) on isolated eyes. Slight corneal swelling (up to 5%) was observed in one eye during the four hour observation period. Corneal opacity (severity 1 or 2) was noted in all eyes and fluorescein retention (severity 0.5) was observed in one eye. No other corneal effect was observed. The negative and positive control group results demonstrate that the study was valid.

Based on this in vitro eye irritation test in the isolated chicken eyes, the test item is not classified as a severe irritant and not classified as non-irritant. An in vivo study was therefore required for classification.

In vivo data

In order to assess the in vivo eye irritation potential of the substance, Matting (2015) conducted a study in accordance with OECD guideline 405. Three rabbits received a single instillation after which they were observed during 72 h. No Initial Pain reaction/pain reaction was observed. One hour after treatment, conjunctival redness (score 1 or 2), chemosis (score 1 or 2) and discharge (score 3) were seen in all rabbits. All symptoms had fully reversed in all animals by the 72 hours observations. Fluorescein staining was negative in all animals at 24, 48 and 72 hours after instillation. The control eyes were symptom-free during the study.

Based on the available in vivo data which is considered to be adequate, reliable and conclusive for eye irritation, the substance is considered to not be an irritant.


Justification for selection of skin irritation / corrosion endpoint:
GLP compliant, guideline study, no restrictions, fully adequate for assessment.

Justification for selection of eye irritation endpoint:
GLP compliant, guideline study, no restrictions, fully adequate for assessment.

Justification for classification or non-classification

Skin Irritation / Corrosion

Two EpiSkin studies were conducted following OECD guidelines and in compliance with GLP Directive 2004/10/EC. Both results are valid, reliable and adequate for the purpose of risk assessment, classification and labelling.

The substance does not fall into the criteria for classification as a corrosive or irritant substance according to Regulation (EC) 1272/2008, Part 3.2., and the substance is not considered to be classified under Directive 2001/59/EEC, 3.2.5 and 3.2.6. The result is conclusive.

Eye Irritation

Results from a validated in vivo study for eye irritation on rabbits, conducted according to OECD Guideline 405 demonstrated the substance to be non-irritating to the eye. The study was carried out in compliance with GLP Directive 2004/10/EC. The conclusion is considered valid, reliable and adequate for the purpose of risk assessment, classification and labelling.

The substance does not fall into the criteria for classification as irritant substance according to Regulation (EC) 1272/2008, Part 3.3.2., and the substance is not considered to be classified under Directive 2001/59/EEC, 3.2.6.2. The result is conclusive.