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Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 February 2013 - 01 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the OECD 406 guideline and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
LLNA proved to be unreliable in predicting skin sensitization potential of diesel that is known to be a non-sensitizer. A valid GPMT inevitably showed that CTO diesel is not a skin sensitizer. More comprehensive justification is provided in the separate RSS "Skin sensitisation. supporting expert report"
Species:
guinea pig
Strain:
other: LAL/HA/BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: LAB-ÁLL Bt. Budapest, 1174 Hunyadi u. 7.
- Age at study initiation: 6 weeks
- Weight at study initiation: 283-371 g
- Housing: in Macrolon cages size IV, with 5 animals / cage
- Diet (e.g. ad libitum): CuniFort Diet for Rabbits (Lot 121217U203 and 130204U111; Bonafarm-Bábolna Takarmány Ltd, Hungary) ad libitum
- Tap water containing 50 mg/100 ml ascorbic acid ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 - 24.0 °C
- Humidity (%): 24 - 57 %
- Air changes (per hr): 15 - 20 air changes / hour
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal and epicutaneous
Vehicle:
other: sunflower oil
Concentration / amount:
Main study I: Intra-dermal induction exposure

Test groups:
A series of three injections was administered on each side of the scapular region of treatment group animals, as follows, resulting in six injections per animal:
- 2 injections with 0.1 ml of Freund's Complete Adjuvant mixed with physiological saline (1:1) (v/v),
- 2 injections with 0.1 ml of the test item in Sunflower oil at 5% (w/v) concentration,
- 2 injections with 0.1 ml of the test item in 5 % (w/v), formulated in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Control group:
The control animals were treated similarly as the test group; however, the vehicle without the test item was used for injections as follows:
- 2 injections with 0.1 ml mix of Freund's Complete Adjuvant and physiological saline (NaCl 0.9 %) (1:1) (v/v),
- 2 injections with 0.1 ml of Sunflower oil,
- 2 injections with 0.1 ml of 50 % formulation of Sunflower oil in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and physiological saline.

Main study II: Dermal Induction Exposure

The test group:
- Animals were treated with approximately 0.5 ml of the 100% (undiluted) test item.

The control group:
- Control animals were treated with Sunflower oil.

Main study III: Challenge Exposure
Test and the control groups.
A 2.5x2.5 cm² patch of sterile gauze was saturated with the test item at 25% (w/v) in Sunflower oil concentration and applied to the left flank of all animals.
The right shaved flank area of all animals was treated with a 50% dilution of the maximum dermal challenge dose (i.e. 12.5 (w/v) % in Sunflower oil).
Route:
epicutaneous, occlusive
Vehicle:
other: sunflower oil
Concentration / amount:
Main study I: Intra-dermal induction exposure

Test groups:
A series of three injections was administered on each side of the scapular region of treatment group animals, as follows, resulting in six injections per animal:
- 2 injections with 0.1 ml of Freund's Complete Adjuvant mixed with physiological saline (1:1) (v/v),
- 2 injections with 0.1 ml of the test item in Sunflower oil at 5% (w/v) concentration,
- 2 injections with 0.1 ml of the test item in 5 % (w/v), formulated in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Control group:
The control animals were treated similarly as the test group; however, the vehicle without the test item was used for injections as follows:
- 2 injections with 0.1 ml mix of Freund's Complete Adjuvant and physiological saline (NaCl 0.9 %) (1:1) (v/v),
- 2 injections with 0.1 ml of Sunflower oil,
- 2 injections with 0.1 ml of 50 % formulation of Sunflower oil in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and physiological saline.

Main study II: Dermal Induction Exposure

The test group:
- Animals were treated with approximately 0.5 ml of the 100% (undiluted) test item.

The control group:
- Control animals were treated with Sunflower oil.

Main study III: Challenge Exposure
Test and the control groups.
A 2.5x2.5 cm² patch of sterile gauze was saturated with the test item at 25% (w/v) in Sunflower oil concentration and applied to the left flank of all animals.
The right shaved flank area of all animals was treated with a 50% dilution of the maximum dermal challenge dose (i.e. 12.5 (w/v) % in Sunflower oil).
No. of animals per dose:
MAIN study I MAIN study II MAIN study III
Dose group Control group Test group Control group Test group Control group Test group
Number of animals 5 10 5 10 5 10
Details on study design:
RANGE FINDING TESTS:
A series of test item concentrations was tested to identify the primary irritation following intra-dermal injection and dermal application: 0.5, 1, 2.5 and 5% (w/v) concentrations were used for intra-dermal injection and 10, 25, 50% (w/v) and 100% (undiluted) for dermal application. Local effects were examined and scored 1, 24, 48 and 72 hours after patch removal. Skin effects were scored for erythema and oedema, any other observations of changes to the skin was recorded.

Two concentrations were injected on the right side and another two concentrations on left side of the animals. Each concentration was injected in duplicate, so each animal received eight injections. Two animals were used per concentration.

It was found that the test item at concentrations of 0.5, 1, 2.5 and 5% (w/v) produced no reaction (scores 0-0) in the skin of guinea pigs after the intra-dermal application.

It was found that 0.5 ml of the test item formulations at concentrations of 100% (undiluted), and 50% (w/v) caused very slight erythema and concentrations of 25 and 10% (w/v) produced no reaction (scores 0-0) on the skin of guinea pigs after the dermal application.

On the basis of results of the Preliminary Dose Range Finding Study, the 5% (w/v) concentration was used for intra-dermal treatment and 100% (undiluted) formulation was used for dermal induction treatment.

MAIN STUDY
A. INDUCTION EXPOSURE
Intra-dermal induction (MAIN study I)

- No. of exposures: 1

- Test groups: 1
A series of three injections was administered on each side of the scapular region of treatment group animals, as follows, resulting in six injections per animal:
* 2 injections with 0.1 ml of Freund's Complete Adjuvant mixed with physiological saline (1:1) (v/v),
* 2 injections with 0.1 ml of the test item in Sunflower oil at 5% (w/v) concentration,
* 2 injections with 0.1 ml of test item in 5 % (w/v), formulated in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

- Control groups: 1
The control animals were treated similarly as the test group; however, the vehicle without the test item was used for injections as follows:
* 2 injections with 0.1 ml mix of Freund's Complete Adjuvant and physiological saline (NaCl 0.9 %) (1:1) (v/v),
* 2 injections with 0.1 ml of Sunflower oil,
* 2 injections with 0.1 ml of 50 % formulation of Sunflower oil in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and physiological saline.

- Site: shoulder

- Duration of exposure: 24 h

- Concentrations: 5 % (w/v)

Dermal induction (MAIN study II)

- No. of exposures: 1

- Test groups: 1
* Animals were treated with approximately 0.5 ml of the 100% (undiluted) test item.

- Control groups: 1
* Control animals were treated with Sunflower oil.

- Site: shoulder

- Duration of exposure: 48 h

- Concentrations: 100 %

B. CHALLENGE EXPOSURE
Two weeks after the topical induction application, the animals were exposed to a dermal challenge dose.
- No. of exposures: 1
- Exposure period: 24 h
- Test groups: 2
- Control group: 2
- Site: Right and left flank
- Concentrations: 25 % and 12.5 % (w/v) in Sunflower oil
- Evaluation (hr after challenge): 24 h and 48 h after the patch removal

OTHER:
Challenge controls:
In the control animals no visible skin reactions were observed following challenge with test item at a concentration of 25 % (w/v) in Sunflower oil at the 24 and 48 hours examination
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole
Positive control results:
Challenge with reference item 2-Mercaptobenzothiazole resulted positive response in test animals sensitised previously. The net response values at the 24 and 48 hours observations represented an incidence rate of 80% and 70% and the net score values of 1.00 and 0.70 respectively. The dermal scores represented discrete or moderate erythema (score 1 or 2) developed on the skin of sensitised guinea pigs.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25 % (w/v)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25 % (w/v). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study..
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25 % (W/v)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25 % (W/v). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study..
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
12.5 % (w/v)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 12.5 % (w/v). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study..
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
12.5 % (w/v)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 12.5 % (w/v). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study..
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50 %
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
no data
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 50 %. No with. + reactions: 8.0. Total no. in groups: 10.0. Clinical observations: no data.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
50 %
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
no data
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 50 %. No with. + reactions: 7.0. Total no. in groups: 10.0. Clinical observations: no data.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Remarks on result:
no indication of skin sensitisation

There were no deaths. No signs of systemic toxicity were noted in the animals during the test. There were no notable differences in bodyweight changes between the test animal group and the control group.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
Sensitisation potential of of test item was studied in the guinea pig using the Magnusson and Kligman Method. Challenge with test item CTO Naphtha evoked no positive responses in the test animals sensitised previously with the test item or with control item. Under the conditions of the present assay the test item was shown to have no sensitisation potential.
Executive summary:

This study was regarded reliable without restrictions since the the study was conducted according to the OECD 406 guideline and in compliance with GLP.

Ten test animals were subjected to sensitisation procedures in a two-stage process, i.e. an intra-dermal treatment and a topical application. The test item was used at a concentration of 5% (w/v) in Sunflower oil for intra-dermal injections and at a concentration of 100% (undiluted) for dermal sensitisation treatment. Two weeks after the last induction exposure, a challenge dose (at a concentration of 25% (w/v) in Sunflower oil) was administered on the left flank of animals. The right flank area of animals was treated with 50% dilution with Sunflower oil of the maximum dermal challenge dose as a safeguard dose (12.5% (w/v) Sunflower oil). Challenge was performed by dermal application of the test item.

Five control guinea pigs were simultaneously exposed to Sunflower oil during the sensitisation phase I (intra-dermal treatment). During the sensitisation phase II (dermal treatment) the control animals were treated with Sunflower oil and they were treated with the test item at a concentration of 25 and 12.5% (w/v) in Sunflower oil only during the challenge.

Test group.

After the challenge with the test item at a concentration of 25% (w/v) in Sunflower oil, no positive response was observed in the treated animals. The mean of the scores was 0.00 according to the 24 and 48-hours results. The right shaved flank area of all animals was treated with a test item concentration of 12.5% (w/v) in Sunflower oil as a safeguard dose and no reaction was noted.

Control group.

After the challenge with the test item at a concentration of 25% (w/v) in Sunflower oil no visible changes were found at the 24 and 48 hours examinations. The right shaved flank area of control animals was treated with a test item concentration of 12.5% (w/v) in Sunflower oil as a safeguard dose and no reaction was noted.

In conclusion, challenge with the test item evoked no positive responses in the test animals sensitised previously with the test item or in the control group. The net response value represented an incidence rate of 0 % and the net score value of 0.00.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Skin sensitisation potential of renewable hydrocarbons of wood origin (naphtha type fraction) was studied using the murine Local Lymph Node Assay (LLNA) and the Guinea Pig Maximisation Test (GPMT). The expert justification for the use of GPMT is provided by Viluksela, M. (2013).

As an easy-to-perform animal test LLNA is the recommended sensitisation assessment method for REACH registration. Contrary to the skin sensitisation data from earlier studies on most biofuels and fossil fuels, the registering substance showed clear positive dose-related responses in LLNA. Chemical analyses on the substance did not identify known sensitizers or other compounds that would explain the positive responses. Furthermore, the performance of LLNA in predicting skin sensitizing potential of chemicals has been recently questioned due to the tendency for false positive results. Because of the documented poor performance of LLNA it was considered necessary to confirm the skin sensitization potential of the test substance using GPMT.

Based on the fact that the LLNA test tends to overestimate the sensitizing potential of fossil fuels resulting in false positive results the LLNA test is considered unreliable. Thus GPMT test is selected as a key study and LLNA test as a supporting study.

GPMT (key)

The dermal sensitisation potential of renewable hydrocarbons (naphtha type fraction) was assessed in a GLP-compliant guideline guinea pig maximisation test (OECD 406) using 5 control animals and 10 test animals (Török-Bathó, M, 2013). On the basis of results of the preliminary testing, the 5 % (w/v) test item concentration in olive oil was used for intradermal injection and 0.5 ml of the 100 % test item was used for dermal induction treatment. Control animals were treated with the sunflower oil.

The challenge exposure was performed two weeks after the induction applications. All animals of the treatment and control group were treated with the 25 % (w/v) concentration as a challenge dose and at concentration of 12.5 % (w/v) as a safeguard dose.

Challenge with the test item evoked no positive responses in the test animals sensitised previously with the test item or with the control item. The net response value represented an incidence rate of 0 % and the net score value of 0.00. Dermal responses were recorded at 1, 24, 48 and 72 hr after the patch removal. No dermal irritation was observed during the observation period in any animal.

The result demonstrates that renewable hydrocarbons of wood origin (naphtha type fraction) is not a skin sensitiser.

LLNA (supporting)

The skin sensitisation potential of renewable hydrocarbons (naphtha type fraction) was assessed using the murine local lymph node assay (OECD 429) (Sanders, A., 2012). The test substance was applied over three consecutive days at three concentrations. Lymph node weights were measured and DPM values were calculated in all treated groups. The SI values over 3 were determined at 100 % concentration. In general, when the SI for any single treatment dose group is ≥ 3, the test item is regarded as a potential skin sensitiser. According to the EC3 value (88 %) calculated for the test substance, the substance should be classified as skin sensitiser according to CLP Regulation 1272/2008.

However, the positive result of this study for renewable hydrocarbons of wood origin (naphtha type fraction), is contrary to the skin sensitisation data from earlier studies on most biofuels and fossil fuel. The performance of LLNA in predicting skin sensitizing potential of chemicals has been recently questioned due to the tendency for false positive results (Kreiling et al., 2008; Ball et al., 2011). These comparative studies consistently indicated that LLNA tends to overestimate the sensitizing potential of chemicals resulting in false positive sensitisation classifications. Skin sensitization studies in guinea pigs using the Magnusson and Kligman method were carried for the test substance at the CiToxLAB Hungary Ltd. in accordance with the OECD, EU and US test method guidance and according to GLP (Török-Bathó, M., 2013). In this study the test substance did not show to have sensitisation potential.


Migrated from Short description of key information:
GPMT (key study): Skin sensitisation was studied using the Guinea Pig Maximisation Test (GPMT) carried for renewable hydrocarbons of wood origin (naphtha type fraction). In this study the test substance was not shown to have sensitisation potential.

LLNA (supporting): The skin sensitisation potential of the substance was assessed using the murine local lymph node assay. The LLNA study result was considered false positive due to the tendency to give positive results to fossil fuels and biofuels.

Justification for selection of skin sensitisation endpoint:
The skin sensitisation study in guinea pigs using the Magnusson and Kligman method (GPMT) is considered to be reliable in predicting sensitisation potential of this substance. This study was conducted according to the OECD 406 guideline and in compliance with GLP.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:
Justification for selection of respiratory sensitisation endpoint:
No respiratory sensitisation studies are conducted for the substance or for the read-across substances. Taking into account the absence of skin sensitization potential of this substance and the lack of sensitisation from occupational exposure settings of read-across substances, respiratory sensitisation is not expected to be of concern for the substance considered in this chemical safety assessment.

Justification for classification or non-classification

Based on the result of the key skin sensitisation study conducted in guinea pigs (GPMT), renewable hydrocarbons of wood origin (naphtha type fraction) is not classified for skin sensitisation according to the CLP Regulation No. 1272/2008 and according to EU Directive 67/548/EEC.

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