Registration Dossier

Administrative data

Description of key information

Skin irritation: 
In this in vitro skin irritation test in the EPISKIN model with test item Tar wood the results indicated that the test item is Irritant (I) [EU: R38; UN GHS: Category 2].
Skin corrosion:
In conclusion, in this in vitro EPISKIN model test with Tar wood the results indicated that the test item is not corrosive to skin.
Eye irritation:
The test item Tar wood is classified as mild eye irritant in the Human Cornea Model test.
Respiratory irritation:
Respiratory irritation caused by tarwood was not studied.

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:
12.02.2014 - 14.02.2014
Reliability:
1 (reliable without restriction)
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EpiSkin™ SOP, Version 1.8 (February 2009),
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Species:
other: not applicable (this is an invitro-test)
Strain:
other: not applicable (this is an invitro-test)
Type of coverage:
other: not applicable (this is an invitro-test)
Preparation of test site:
other: not applicable (this is an invitro-test)
Vehicle:
not specified
Remarks:
not applicable (this is an invitro-test)
Controls:
other: not applicable (this is an invitro-test)
Duration of treatment / exposure:
15 minutes exposure and 42 hours post incubation
Observation period:
42 hours
Number of animals:
not applicable (this is an invitro-test)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Episkin SM
Value:
< 50
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

see study report for more information

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Remarks:
Migrated information
Conclusions:
In this in vitro skin irritation test in the EPISKIN model with test item Tar wood the results indicated that the test item is Irritant (I) [EU: R38; UN GHS: Category 2].
Executive summary:

EpiSkinTMSM test of Tar wood has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 26 July 2013.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

The test item has an intrinsic colour (brown), one additional chemical-treated tissue was used for the non-specific OD evaluation.SDS (5% aq.) and PBS treated epidermis were used as positive and negative controls respectively (three units / control). For each treated tissue viability was expressed as a percentage relative to negative control.

The test item is considered to be irritant to skin, 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 item showed significantly reduced cell viability in comparison to the negative control (relative mean value: 13 %). All obtained test item viability results were below 50 % when compared to the viability values obtained from the negative control, therefore the test item was considered to be irritant to skin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
27.02.2014
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
other: Ocular Irritation Protocol: Neat Method (MTT ET-50) for use with EpiOcular TM Tissues, Rev: 17. Jan. 2012 by MatTek.
Deviations:
no
Principles of method if other than guideline:
SOP 118 00 890, edition 2 dated 01. Aug. 2013 ,“Bestimmung der hornhautreizenden Eigenschaften (Ermittlung ET50) mit EpiOcularTM“Additional: Stern M., Klausner M., Alvarado R., Renskers K., Dickens M., 1998. Evaluation of the EpiOcular Tissue Model as an Alternative to the Draize Eye Irritation Test. Toxicology in Vitro 12, 455-461
GLP compliance:
yes (incl. certificate)
Species:
other: three-dimensional human cornea model tissue
Strain:
other: not applicable (this is an invitro test)
Vehicle:
other: not applicable (this is an invitro test)
Controls:
other: not applicable
Duration of treatment / exposure:
3 min., 30 min. and 60 min.
Number of animals or in vitro replicates:
not applicable
Irritation parameter:
other: ET50
Run / experiment:
EpiOcular Test
Value:
32.63
Negative controls validity:
valid
Positive controls validity:
valid

The test item showed the ability of direct Formazan reduction. Therefore, a functional test with freeze-killed tissues was performed and all measured values were corrected by the net formazan reduction of the freeze-killed tissues.

After 3 minutes treatment with the test item, the corrected relative absorbance values were reduced to 63.2 %. After 30 minutes treatment, corrected relative absorbance values were reduced to 57.5%. And after 60 minutes treatment, the corrected relative absorbance values were reduced to 31.9%.

From these values, the ET50was calculated mathematically. The calculated ET50of the test item Tar wood was 38.82 min.

The test item is considered as mild eye irritant.

The criterion for optical density of the negative control was fulfilled: The optical density of 1.746 is in the range of the historical data.

The positive control induced a decrease in the relative absorbance as compared to the negative control to 75.3 % for the 45 minutes treatment interval and 32.2 % for the 15 minutes treatment interval. The calculated ET50was 32.63 min., thus ensuring the validity of the test system.

Interpretation of results:
other: mild irritant
Remarks:
Criteria used for interpretation of results: other: see table 7.5-a in the final report
Conclusions:
Tar wood is classified as mild eye irritant in the Human Cornea Model test.
Executive summary:

In the pre-test, the test item Tar wood showed the ability of direct formazan reduction. Therefore, a functional check with freeze killed tissues was performed as well.

In the main study, the test item Tar wood was applied to a three-dimensional human cornea model tissue for three different exposure times in duplicate (3 min., 30 min. and 60 min.).

100 µL of the liquid test item were applied to each tissue.

Demin. water was used as negative control, 0.3% Triton X 100 solution was used as positive control.

After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT which can be reduced to a blue formazan. Formazan production was measured by measuring the optical density (OD) of the resulting solution.

After treatment with the negative control, the absorbance values were well within the historical range and showed no cytotoxic effects. The positive control showed clear irritating effects for both treatment intervals.

After 3 minutes treatment with the test item, the relative absorbance values were reduced to 63.2 %. After 30 minutes treatment, relative absorbance values were reduced to 57.7 %. And after 60 minutes treatment, the relative absorbance values were reduced to 31.9 %. These values were calculated after subtraction of direct formazan reduction.

From these values, the ET50was calculated mathematically. The calculated ET50of the test item Tar wood was 38.82 min.

Therefore, the test item Tar wood is classified as mild eye irritant in the Human Cornea Model test.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

The test item has an intrinsic colour (brown), one additional chemical-treated tissue was used for the non-specific OD evaluation.SDS (5% aq.) and PBS treated epidermis were used as positive and negative controls respectively (three units / control). For each treated tissue viability was expressed as a percentage relative to negative control.

The test item is considered to be irritant to skin, 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 item showed significantly reduced cell viability in comparison to the negative control (relative mean value: 13 %). All obtained test item viability results were below 50 % when compared to the viability values obtained from the negative control, therefore the test item was considered to be irritant to skin.

Skin corrosion:Disks of EPISKIN (two units / chemical) were treated with test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of 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 precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls respectively. Since the test item is a coloured material, in addition to the normal procedure two additional chemical-treated tissues (as additional control) were used for the non-specific OD evaluation to detect and correct the test item interference with the viability measurement in order to avoid a false estimation of viability. For each treated tissue, viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control. The test item did not showed significantly reduced cell viability in comparison to the negative control after four hours of exposure. Both individual tissue viabilities were above 35 % of the mean negative control value. The average test item treated tissue viability was 119 %. In conclusion, in this in vitro EPISKIN model test with Tar wood the results indicated that the test item is not corrosive to skin.

Justification for selection of eye irritation endpoint:
Not available.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: slightly irritating

Justification for classification or non-classification

Skin irritation: The test substance is considered to be irritant to skin (R38), 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 prediction model (PM) is described below:

Criteria for In Vitro interpretation

Classi

fication

EU

UN GHS

Mean tissue viability % is ≤ 50 %

Irritant (I) R38

Category 2

Mean tissue viability % is > 50 %

Non Irritant (NI)

No Category

Skin corrosion:

The prediction model below corresponds to the methods agreed by EU regulatory agencies in line with OECD 431 (OECD, 2004). The cut-off value of 35 % and classification method was validated in an international validation of this kit (Fentem, 1998).The interpretation of test results is summarized in the following table:

Classification

Packing group / classification categories

Criteria for In Vitro interpretation

UN

Corrosive: 

Optional Sub- category 1A

Mean tissue viability is < 35 % after 3 min exposure

Corrosive: 

Optional Sub- category 1B and 1C

Mean tissue viability is ≥ 35 % after 3 min exposure and < 35 % after 1 hour exposureOR

Mean tissue viability is ≥ 35 % after 1 hour exposure and < 35 % after 4 hours exposure

Non corrosive

Mean tissue viability is ≥ 35 % 4 hours exposure

EU (67/548/EEC)

Corrosive class R35

Mean tissue viability is < 35 % after 3 min exposure

Corrosive class R34

Mean tissue viability is ≥ 35 % after 3 min exposure and < 35 % after 4 hours exposure

Non corrosive

Mean tissue viability is ≥ 35 % 4 hours exposure

EU 

(EC n° 1272/2008 CLP)

Corrosive 1A

Mean tissue viability is < 35 % after 3 min exposure

Corrosive: 1B and 1C

Mean tissue viability is ≥ 35 % after 3 min exposure and < 35 % after 1 hour exposureOR

Mean tissue viability is ≥ 35 % after 1 hour exposure and < 35 % after 4 hours exposure

Non corrosive

Mean tissue viability is ≥ 35 % 4 hours exposure

Eye irritation:

The lower the ET50value, the higher is the eye irritation potential of the test item.

Eye irritation is assessed using the criteria given in the following table:

Classification

ET50-Range (minutes of exposure)

non/minimal irritant

> 60

mild

31 - 60

moderate

3 - 30

severe/extreme

< 3