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Administrative data

Description of key information

Skin irritation: In vitro: Irritating (renewable hydrocarbons of wood origin (naphtha type fraction))
In vivo: Irritating (read-across substance; unleaded gasoline)
Eye irritation: In vitro: Not irritating (renewable hydrocarbons of wood origin (naphtha type fraction))

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:
weight of evidence
Study period:
04 April 2012 - 17 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is carried out based on the OECD No. 439 "In Vitro Skin irritation" and in compliance with OECD principles of Good Laboratory Paractice (GLP).
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes
Species:
human
Strain:
other: epidermal keratinocytes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 microliters
Duration of treatment / exposure:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes.
Observation period:
Post-exposure incubation period was 42 hours.
Details on study design:
Pre-incubation: Each epidermis unit was transferred into maintenance medium filled wells. The tissues were incubated at 37 deg. C, 5% CO2 in air overnight

Application of the test item: Triplicate tissues were treated with the test item for an exposure period of 15 minutes. 10 microliters were applied to the epidermis surface. Negative control epidermis units were treated with DPBS and positive control epidermis units were treated with 5% SDS (w/v) solution.

Washing: At the end of the 15 min exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca2+ and Mg 2+. The sinsed tissues were transferred to the second column containg 2 ml of maintenance medium. The rinsed tissues were incubated at 37 deg. C, 5% CO2 in air for 42 hours.

Cell viability measurements:Following the 42-hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 min to homogenise the released inflammatory mediators in the maintanance medium. 1.6 ml of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. The MTT assay is a validated quantitative method which should be used to measure cell viability. The tissue is placed in Formazan solution for 3 hours. The precipitated blue formazan product is then extracted from the tissue using acidic isopropanol, and the concentration of formazan is measured by determining the OD at 570 nm.


SCORING SYSTEM:
Classification of irritation potential is based upon relative mean tissue viability following the 15min exposure period followed by the 42 hour post exposure incubation period.
Criteria for in vitro interpretation Classification
Relative mean tissue viability is ≤ 50% Irritant
Relative mean tissue viability is > 50% Non-Irritant
Irritation / corrosion parameter:
other: other: The relative mean viability (%) of the treated tissues
Value:
13.3
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15min. Max. score: 100.0. Remarks: viability 0-50% is classified as an irritant; viability > 50 % is classified as a non-irritant . (migrated information)
Irritant / corrosive response data:
See table 1 in attached background material.
Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
The skin irritation potential of the test item was evaluated using EPISKIN TM reconstructed human epidermis model. Based on the study result the the test item is classified as irritant to the skin.
Executive summary:

The study was regarded reliable without restrictions, since the study is carried out based on the OECD No. 439 "In Vitro Skin irritation" and in compliance with OECD principles of Good Laboratory Paractice (GLP).

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to micro tubes and stored in freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containg acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the extraction period each tube was mixed and duplicate samples were measured for optical density at 540nm. The individual and mean OD540 values, standard deviations and tissue viabilities for the test item, negative control item, and positive control item were calculated. The relative mean viability of the test item treated tissues was 13.3 % after a 15 min exposure period.

This study is used as WoE in the hazard assessment. Based on the study results the classification of this substance is Skin Irrit. 2.

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:
weight of evidence
Study period:
15 May 2012 - 17 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: At present there are no validated or OECD adopted in vitro tests for eye irritation. However, the test used here, SkinEthic™ HCE (SkinEthic, France), was conducted in accordance with GLP standards and the study is well documented.
Qualifier:
no guideline available
Principles of method if other than guideline:
The test is based on the SkinEthic HCE model (SkinEthic Laboratories, Nice, France). Triplicate tissues were treated with the test item for an exposure period of 10 minutes. At the end of the exposure period each tissue was taken for MTT assay and optical density at 540nm to measure cell viability were determined. The individual and mean OD540 values and tissue viabilities for the test item, negative control item, and positive control item were calculated.
GLP compliance:
yes
Species:
other: Human corneal epithelium
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 30 microliters
Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
See details on study details
Number of animals or in vitro replicates:
Not used. In vitro test.
Details on study design:
Triplicate tissues were treated with 30 microliters of the test item for 10 minutes. Triplicate tissues were treated with positive and negative control solutions. At the end of the exposure period each tissue was rinsed with PBS without Ca/Mg. Following rinsing, the tissues (two per group) were transferred to a pre-labelled 24-well plate containing 300 microliters of a 0.5mg/ml MTT solution. The plate was placed into an incubator for three hours. At the end of the incubation period the tissue inserts were rinsed twice with PBS and transferred to a new pre-labelled 24-well plate containing 0.75 ml of isopropanol in each well. An extra 0.75 ml of isopropanol was added onto each tissue and the plate was seled to prevent isopropanol evaporation. The plate was allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue. At the end of the extraction period, the solution in each tissue insert were homogenized and measured for optical density at 540nm.
Irritation parameter:
other: Relative mean viability
Run / experiment:
mean
Value:
97.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
The eye irritation potential of the test item was evaluated using SkinEthic HCE reconstructed human corneal epithelium model. Based on the study result the the test item is considered to be a non-irritant.
Executive summary:

The study was regarded reliable without restrictions, since the study is carried out in compliance with OECD principles of Good Laboratory Paractice (GLP) and the documentation is sufficient for assessment. However, at present the method is not validated in vitro tests for assessment of eye irritation.

Triplicate tissues were treated with the test item for an exposure period of 10 minutes. At the end of the exposure period each tissue was taken for MTT-loading. After MTT loading tissue inserts were placed into isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the extraction period each well was mixed and samples were measured for optical density at 540nm. The individual and mean OD540 values and tissue viabilities for the test item, negative control item, and positive control item were calculated. The relative mean viability of the test item treated tissues was 97.1 % after a 10 min exposure period. Based on this data test item is considered to be a non-irritant.

This study is used as key study in the hazard assessment.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

In vivo testing is not scientifically justified since the substance is classified as a skin irritant based on the read-across data available for fossil diesel fuel. The reliable in vitro skin irritation results of the substance also indicated positive irritation. The read-across data on fossil fuel and the in vitro study results conducted for the substance itself were used as WoE for this endpoint.

In vitro study conducted for the substance was used as WoE to evaluate the skin irritation properties of renewable hydrocarbons of wood origin (naphtha type fraction) Warren, (2012). The information on skin irritation from the in vivo skin irritation study for the read-across substance supported the conclusions derived based on the in vitro study (API, 1980).

In vitro skin irritation study was carried out based on the OECD 439 guideline and in compliance with OECD principles of Good Laboratory Practice (GLP) (Warren, N., 2012). The test was used to evaluate the skin irritation potential of the substance using EPISKIN TM reconstituted human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of assay was based on the cytotoxicity in human epidermal cultures following topical exposure of the test item by means of colourimetric MTT reduction assay. The relative mean viability of the test item treated tissues was 13.3 % after a 15 min exposure period. Based on this value renewable hydrocarbons of wood origin (naphtha type fraction) was considered to be irritant to the skin.

This UVCB substance is a complex mixture of hydrocarbons. When there is no in vivo data on the substance itself the read-across data was also used to avoid unnecessary animal testing. The read-across from fossil unleaded gasoline is justified because the toxicokinetics and toxicological properties of renewable gasoline are considered similar to fossil gasoline based on the similar composition and physical-chemical properties. The read-across justification and the data matrices are presented in the annex 1 of the CSR.

Since there is no in vivo data available from the substance, the skin irritation potential is evaluated from published data available from read-across substance. API (1995) had conducted in vivo skin irritation study for the similar substance, unleaded gasoline. In this study New Zealand White rabbits were exposed unleaded gasoline for 24 hours in occlusive conditions. The mean erythema and oedema scores during the 24 to 72 hour observation periods were 2.56 and 1.89, respectively. According to the conditions of this study, the test substance is considered a skin irritant.

Based on the in vitro study from the test substance and in vivo skin irritation study result conducted for the read-across substance (unleaded gasoline), renewable hydrocarbons of wood origin (naphtha type fraction) will be classified for skin irritation.

 

Eye irritation

Weight of evidence approach was used to evaluate the testing needs of this endpoint. Reliable in vitro eye irritation study was conducted for the renewable hydrocarbons of wood origin (naphtha type fraction) (Warren, N., 2012). Further evidence on eye irritation is derived from the in vivo eye irritation data from gasolines generally.

In vitro eye irritation study was carried out using the SkinEthic reconstructed Human Corneal Epithelium Model (HCE, SkinEthic Laboratories, France) and in compliance with OECD principles of Good Laboratory Practice (GLP) (Warren, N., 2012). The principle of an assay was based on the cytotoxicity in human cornel epithelial cultures following 10 minutes treatment of the test item by means of colourimetric MTT reduction assay. The relative mean viability of the test item treated tissues was 97.1 %. Based on this value the test item was considered to be non-irritant to the eye.

Generally, there is no clear evidence on eye irritation derived from read-across substances, gasoline and naphtha stocks, when instilled into rabbit eye. The effects seen have been very mild and no clear dose response has been observed. For example, naphtha, (petroleum), hydrotreated light composed of predominantly C7 to C9 paraffins did not cause eye irritation in rabbits.

Based on the general data on gasolines from animal studies and in vitro data on the substance no classification is warranted for eye irritation.


Justification for selection of skin irritation / corrosion endpoint:
The reliable in vitro study conducted for the substance.

Justification for selection of eye irritation endpoint:
The reliable in vitro study conducted for the substance.

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

Based on the in vitro skin irritation information data from renewable hydrocarbons (naphtha type fraction) and from in vivo data from surrogate fossil fuels, the substance has to be classified for Skin Irrit. 2 H315 according to CLP Regulation 1272/2008 and for Xi; 38 according to Directive 67/548/EEC.

Based on the in vitro eye irritation information from the substance itself and on in vivo eye irritation information from surrogate fossil fuels generally, renewable hydrocarbons (naphtha type fraction) has not to be classified for eye irritation according to CLP Regulation 1272/2008 and Directive 67/548/EEC.