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Administrative data

Description of key information

Skin (OECD 439): in vitro EPISKIN test: not irritating
Skin (OECD 404) on analogue: rabbit, not irritating
Eye (OECD 437): in vitro BCOP: not causing serious eye damage
Eyes: (OECD 405) on analogue, rabbit: severe irritant

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:
09-04-2014 to 14-04-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study performed according to the guideline and under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Principles of method if other than guideline:
exposure period 15 minutes with post-exposure incubation period of 42 hours
GLP compliance:
yes (incl. QA statement)
Details on study design:
The EpiSKIN assay uses reconstructed human epidermis to assess skin irritation. For the viability test enzymatic conversion of MTT to formazan is used.

In a pre-test direct interaction of the test substance with the detection chemical MTT is assessed.
In the main study triplicate tissues were treated with 30 uL of the test substance during 15 minutes. In addition a negative control (DPBS) and a positive control (5% SDS) were included. After treatment tissues were rinsed, incubated in maintenance fluid for 42 hours at 37 °C and 5% CO2 and thereafter treated with MTT during 3 hours (at 37 °C and 5% CO2). After MTT loading a total biopsy of each epidermis (epidermis and collagen) was made, that was suspended in acidified iso-propanol for 3 days and kept in the refrigerator at 1-10 °C. Thereafter the optical density of the extracted MTT solutions was measured at 562 nm (Anthos 2001 microplate reader). Viability was expressed as percentage MTT conversion versus negative control.
Tissue viability > 50% means non-irritant
Tissue viability ≤ 50% means irritant (Category 2)
Other effects:
The test substance did not interfere with the reduction of MTT to blue formazan salt.

Mean OD562± SD

Relative mean viability ± SD

Negative control:

0.903 ± 0.013

100 ± 1.4%

Test substance:

0.823 ± 0.063

91.2 ± 7.0%

SDS:

0.055 ± 0.008

6.1 ± 0.9%

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: tissue viability > 50%: non-irritant
Conclusions:
The test substance is a non-irritant in the EPISKIN reconstructed human epidermal model.
Executive summary:

In this assay triplicates of reconstructed human skin tissues were treated with 30 uL of the test substance during 15 minutes. After treatment tissues were rinsed, kept for 42 hours and treated with MTT. Optical density of extracted MTT solutions was measured at 540 nm. Viability was expressed as percentage MTT conversion versus negative control. The viability was 91.2%, which is indicative for non-irritant substances.

The test substance is a non-irritant in the EPISKIN reconstructed human epidermal model.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13-05-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study according to the guideline under GLP. Study does not allow definitive conclusion
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
GLP compliance:
yes (incl. QA statement)
Vehicle:
other: 0.9% w/v sodium chloride solution
Amount / concentration applied:
Test item was formulated as a 20% solution in 0.9% w/v sodium chloride.
Positive control: Imidazoleas a 20% w/v solution in 0.9% w/v sodium chloride
Negative control: vehicle
Duration of treatment / exposure:
240 min
Details on study design:
Bovine corneas were obtained from a local abatoir, preserved in HBBS and prepared on arrival in the laboratory. Corneas free of damage were mounted in a closed BCOP holder. Both chambers were filled with MEM and incubated for 60 minutes at 32 ± 1 ºC.
In the test undamaged corneas (3/treatment) were checked for opacity (zero measurement) and exposed to 0.75 mL the test substance, negative or positive control (anterior chamber) at 32 ± 1 ºC for 240 minutes. After exposure the chambers were filled with fresh MEM and opacity was assessed. Corneas were checked visually. In additon the cornes were exposed to sodium fluorin (5 mg/L) (anterior chamber) for 90 minuted. Optical density at 492 nm of the medium in the posterior chamber was measured to assess permeability.
Measurements for opacity were corrected for the initial opacity and the opacity measured in the vehicle controls.
Measurements for permeability were corrected for vehicle control.
All results were averaged.
Irritation parameter:
other: IVIS
Score:
6.1
Remarks on result:
other: mean opacity value + (15 x mean OD492 value)

Treatment

Cornea appearance

(Corrected) opacity value

(Corrected) OD value

In vitro irritancy score

Negative control

clear

3.3

0.042

4.0

Positive control

cloudy

59.3

1.519

82.1

Test substance

clear

4.7

0.093

6.1

 

Interpretation of results:
other: not causing serious eye damage
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The outcome of the BCOP test with the substance shows that the substance is not causing serious eye damage
Executive summary:

The substance is tested in the in vitro BCOP assay. The calculated IVIS based on assessments of opacity and permeability for the test substance is 6.1. This does not allow a definitive conclusion on the irritant properties of the test substance, but indicates that the substance is not causing serious eye damage.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

In an in vitro assay triplicates of reconstructed human skin tissues were treated with 30 uL of the test substance during 15 minutes (Harlan 2014). After treatment tissues were rinsed, kept for 42 hours and treated with MTT. Optical density of extracted MTT solutions was measured at 540 nm. Viability was expressed as percentage MTT conversion versus negative control. The viability was 91.2%, which is indicative for non-irritant substances.

The test substance is a non-irritant in the EPISKIN reconstructed human epidermal model.

In addition data on skin irritation by the analogue and hydrolysis product rosin, fumarated were available. In a primary dermal irritation study performed according to GLP and OECD 404, six young adult New Zealand white rabbits were exposed to 0.5 g of Rosin, fumarated applied for 4 hours under semi-occluded contact to intact skin (Life Science Research, 1991). Animals were observed for a period of 72 hours post-treatment and irritation was scored. Mean erythema and edema scores for 24 to 72 hours were observed 0 in intact rabbit skin. Based on these findings, rosin,fumarated was not considered to be a skin irritant to rabbits, and presents a low skin irritation hazard.

 

Eye irritation

The substance was tested in the in vitro BCOP assay (Harlan 2014). The calculated IVIS based on assessments of opacity and permeability for the test substance was 6.1. This does not allow a definitive conclusion on the irritant properties of the test substance, but indicates that the substance is not causing serious eye damage.

Eye irritation of the analogue and hydrolysis product rosin, fumarated was evaluated in a study performed according to GLP and OECD 405, where 0.1 g was instilled into the eye of one New Zealand White rabbit (Life Science Research, 1991).The animal was observed and then terminated 5 hours after application.After 5 hours, the substance was found to be a severe eye irritant, and further testing was therefore not warranted. Due to the structural similarities, some irritation of the eyes could be expected for resin acids and rosin acids, fumarated, compds. with triethanolamine.

 

Respiratory Irritation

No studies were identified, however, the low vapour pressure indicates that exposure via this route is unlikely.


Justification for selection of skin irritation / corrosion endpoint:
guideline study under GLP

Justification for selection of eye irritation endpoint:
Reliable study. No definitive conclusion can be drawn. The substance is shown not to cause serious eye damage

Effects on eye irritation: irritating

Justification for classification or non-classification

The available data on skin irritation of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.

However, the available data on eye irritation of the test substance do not allow a definitive conclusion on eye irritation. However, in a worst case approach it is concluded that the substance meets the criteria for classification as H319 (causes serious eye irritation Category 2) according to Regulation (EC) 1272/2008 and R36 according to Directive 67/548/EEC.