Registration Dossier

Administrative data

Description of key information

Skin irritation:
The test material is considered not irritating to skin.
Eye irritation:
The test material is considered not irritating to the eye.

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 April to May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
human
Strain:
other: EPISKIN MODEL
Details on test animals or test system and environmental conditions:
Study was completed using the EPISKIN MODEL.
The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes 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 a 13-day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Type of coverage:
open
Preparation of test site:
other: The tissues were incubated at 37°C, 5% CO2 in air overnight.
Vehicle:
water
Controls:
other: Positive and negative controls
Amount / concentration applied:
The test material was ground before use. No vehicle used. Approximately 10 mg of the test material was applied to the epidermis surface.

Duration of treatment / exposure:
15 minutes
Observation period:
6 days
Number of animals:
Triplicate tissues
Details on study design:
The MTT assay, a colourimetric method of determining cell viability is based on reduction of the yellow tetrazolium salt to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.
Day 1-2:
2 ml of maintenance medium, warmed to approximately 37°C, was pipetted into the second column of 3 wells of the 12-well plate.
Triplicate tissues were treated with the test material for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. Approximately 10 mg of the test material was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 µl of sterile distilled water to improve contact between the solid test material and the epidermis. Triplicate tissues treated with 10 µl of DPBS served as the negative control and triplicate tissues treated with 10 µl of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip, taking particular care to cover the centre. After 7-minutes contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period. The plate(s) were kept in the biological safety cabinet at room temperature for 15 minutes.
At the end of the exposure period the tissue was removed and rinsed. The rinsed tissues were incubated at 37°C, 5% CO2 in air for 42 hours.
Day 3-5:
Following the 42-hour post-exposure incubation each 12-well plate was placed onto a plate shaker for 15 minutes to homogenise the released mediators in the maintenance medium. 1.6 ml of the maintenance medium from beneath each tissue was transferred to the pre-labelled micro tubes and stored in a freezer at - 14 to - 30°C for possible inflammatory mediator determination.
2 ml of a 0.3 mg/ml MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12 well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37°C, 5% CO2 in air. At the end of the 3-hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts placed onto labelled 1.5 ml micro tubes containing 500 µl acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
Day 6:
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.
For each tissue, duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µl of acidified isopropanol alone was added to the two wells designated as blanks. The optical density was measured (quantitative viability analysis) at 540 nm.
REMOVAL OF TEST SUBSTANCE-
Washing: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++.
- Time after start of exposure: 15 minutes
SCORING SYSTEM:
Quantitative MTT Assessment (percentage tissue viability). For the test material the relative mean tissue viabilities obtained after the 15-minute exposure period 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:
Relative mean viability (%) = (mean OD540 of test item / mean OD540 of negative control) x100
Classification of irritation potential is based upon relative mean tissue viability following the 15-minute exposure period followed by the 42-hour post-exposure incubation period according to the following:
Relative mean tissue viability is <=50% = Irritant (I) R38
Relative mean tissue viability is >50% = Non-Irritant.
TEST SITE- Area of exposure:- % coverage:
- Type of wrap if used:
Irritation / corrosion parameter:
other: other: Mean OD540
Value:
0.926
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes. Max. score: 0.982. Remarks: EPISKIN MODEL. (migrated information)
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In an OECD 439 study, conducted according to GLP, the relative mean viability of the test material with the treated tissues (using the EPISKIN system, in vitro) was 109.2% after a 15-Minute exposure, therefore, is considered not irritating to skin.
Executive summary:

The relative mean tissue viability for the positive control treated tissues was 8.9% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.7%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 0.848 and the standard deviation value of the percentage viability was 3.2%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 6.0%. The test material acceptance criterion was therefore satisfied.

The relative mean viability of the test material treated tissues was 109.2% after a 15-Minute exposure period following quantitatively correction of the results for direct reduction of MTT.

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:
June - July 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute 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 UK.
- Age at study initiation: Twelve to twenty weeks.
- Weight at study initiation: 2.47 and 2.61 kg
- Housing: The animals were individually housed in suspended cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 °C.
- Humidity (%): 30 to 70%
- Air changes (per hr): Fifteen changes per hour.
- Photoperiod (hrs dark / hrs light): Twelve hours continuous light, twelve hours darkness.
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL- Amount(s) applied: 0.1mL (approximately 57 mg).
For the purpose of the study the test material was ground to a powder before use. The absorption of the test material was not determined.
The test material was placed into the conjunctival sac of the right eye. The left eye remained untreated and was used for control purposes.
Duration of treatment / exposure:
72 hours.
Observation period (in vivo):
72 hours.
Number of animals or in vitro replicates:
Three.
Details on study design:
SCORING SYSTEM:
A modified version of the system described by Kay J.H and Calandra J.C (1962) was used to classify the ocular irritancy potential of the test item.

Irritation parameter:
maximum mean total score (MMTS)
Basis:
mean
Time point:
other: 24h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 48 hours
Irritant / corrosive response data:
No corneal or irisial effects were noted during the study.
Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in two treated eyes one hour after treatment with minimal conjunctival irritation noted in all treated eyes. At the 24-hour observation.
All treated eyes appeared normal at the 48-hour observation.
Other effects:
All animals showed expected gain in body weight during the study.
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material produced a maximum mean score of 2 after 24 hours, fully reversible after 48 hours, therefore, is considered not irritating to the eye.
Executive summary:

In an OECD 405 study, conducted according to GLP, the test material produced a maximum mean score of 2 after 24 hours in male rabbits (fully reversible after 48 hours), and therefore is considered not irritating to the eye.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The registration of the test material employs a combined approach for addressing the required endpoints. When data are not available, data from the read across substances are used to address the required endpoints.

 

The read across substances are structurally similar to registered substance. The substance and the proposed structural analogues are predominantly C32 – C36 alkyl fatty amides. These substances are expected to be metabolised by fatty acid amide hydrolase and esterases. Any differences among the substance structures are not expected to result in any significant differences in the toxicological effects or degradation products.

 

The following endpoints include supporting studies to provide evidence of read across suitability:

  • In vivo skin irritation:

Skin:

Key Study:

In an OECD 439 study, conducted according to GLP, the relative mean viability of the test material with the treated tissues (using the EPISKIN system, in vitro) was 109.2% after a 15-Minute exposure, therefore, is considered not irritating to skin.

 

Supporting Study:

In an OECD 404 study, conducted according to GLP, the read across substances are considered not irritating to skin.

 

Eye

Key Study:

In an OECD 405 study, conducted according to GLP, the test material produced a maximum mean score of 2 after 24 hours in male rabbits (fully reversible after 48 hours), and therefore is considered not irritating to the eye.

 

Supporting Study:

In an OECD 437 study, conducted according to GLP, the ocular irritancy potential of the test material, to the isolated bovine cornea induced an in vitro Irritancy Score of 1.1, and is considered not irritating to the eye.


Justification for selection of skin irritation / corrosion endpoint:
In vitro study conducted according to OECD Guideline under GLP conditions for the test material.

Based upon the results of the in vitro study, a read across in vivo skin irritation (not irritating) and a read across acute dermal toxicity (LD50) of >2,000 mg/kg for the read across substance in vivo skin irritation testing on the test material is considered unnecessary.

Justification for selection of eye irritation endpoint:
In vivo study conducted according to OECD Guideline under GLP conditions.

Justification for classification or non-classification

Skin irritation:

In an in vitro skin irritation study, the relative mean viability of the test material with the treated tissues (using the EPISKIN system, in vitro) was 109.2% after a 15-Minute exposure. Under OECD 439, the substance is considered irritating to the skin if the relative mean tissue viability is <=50% using the EPISKIN system.

While negative in vitro data cannot be considered conclusive for a non - classification, according to Regulation (EC) No 1907/2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) Annex VIII Column 2 8.1.1, an in vivo study does not need to be conducted if an acute toxicity study by the dermal route does not indicate skin irritation up to the limit dose level (2,000 mg/kg bw). In a read across dermal toxicity study very slight erythema was noted (score 1) in 4 animals, with no signs of dermal irritation noted at the test sites of the remaining animals. In addition, in a read across in vivo skin irritation study, another read across substance is considered not irritating to skin.

According to Regulation (EC) No 1272/2008 on the classification, labelling and packaging of substances and mixtures (CLP), a substance is irritating to skin if the mean value is ≥ 2 in at least 2 of 3 tested animals.

Based on the above information, the test material is, therefore, considered to be not classified for skin irritation.

Eye irritation:

In an in vivo study, the test material produced a maximum mean score of 2 after 24 hours in male rabbits (fully reversible after 48 hours).

According to Regulation (EC) No 1272/2008 on the classification, labelling and packaging of substances and mixtures (CLP), a substance is irritating to the eye if the mean value is ≥ 2 in at least 2 of 3 tested animals after 72 hours.

The test material is, therefore, considered to be not classified for eye irritation.