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Toxicological information

Skin sensitisation

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Skin sensitization potential of crude tall oil (CTO) based diesel and naphtha were studied for REACH registration using the murine Local Lymph Node Assay (LLNA). As an easy-to-perform refinement animal test LLNA is the recommended sensitization assessment method for REACH registration. Contrary to the skin sensitization data from earlier studies on most biofuels and fossil fuels, both CTO diesel and CTO naphtha showed clear positive dose-related responses in LLNA. Chemical analyses on CTO diesel and CTO naphtha did not identify known sensitizers or other compounds that would explain the positive responses.
The performance of LLNA in predicting skin sensitizing potential of chemicals has been recently questioned due to the tendency for false positive results. Skin sensitizing properties of a number of chemically diverse organic compounds were compared in LLNA and in the Guinea Pig Maximation Test (GPMT) (Kreiling et al., 2008; Ball et al., 2011). As sensitive and reliable method GPMT (Magnusson and Kligman, 1969) is considered as the benchmark procedure for sensitization testing. It has been successfully used for predicting human sensitizers for more than 40 years. Both comparative studies consistently indicated that LLNA tends to overestimate the sensitizing potential of chemicals resulting in false positive sensitization classifications. LLNA turned out to be positive also for endogenous compounds of the human body and commonly and widely used non-sensitizing constituents of food and cosmetics (Kreiling et al., 2008). Because of the documented poor performance of LLNA it was considered necessary to confirm the skin sensitization potential of CTO diesel and CTO naphtha using GPMT.In addition, for comparative purposes the skin sensitization potential of the regular diesel oil that is known to be a non-sensitizer, was tested with LLNA (Váliczkó, 2013).

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): CTO Diesel
- Substance type: UVCB substance
- Physical state: liquid
- Analytical purity: 100%; UVCB substance
- Lot/batch No.: 12-06947-019
- Expiration date of the lot/batch: 15 December 2013
- Storage condition of test material: room temparature (15-25 deg. C, below 70 RH%)

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: LAL/HA/BR
Sex:
male
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: 271-356 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 - 23.2 °C
- Humidity (%): 24 - 47 %
- Air changes (per hr): 15 - 20 air changes / hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
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

The test and control groups:
A 2.5x2.5 cm² patch of sterile gauze was saturated with the test item at 50 % (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. 25 (w/v) % in Sunflower oil).
Challengeopen allclose all
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

The test and control groups:
A 2.5x2.5 cm² patch of sterile gauze was saturated with the test item at 50 % (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. 25 (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 concentration of 100% (undiluted) caused very slight erythema and dry skin. At concentrations of 50, 25 and 10% (w/v) produced no reaction (scores 0-0) on the skin of guinea pigs.

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-derma 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

Results and discussion

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.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50 % (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: 50 % (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 group
Dose level:
50 % (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: 50 % (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 group
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 group
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:
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.
Key result
Reading:
1st reading
Group:
negative control
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
Sensitisation potential of of the test item was studied in the guinea pig using the Magnusson and Kligman Method. Challenge with test item CTO Diesel 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 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 50 % (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 (25% (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 50 and 25 % (w/v) in Sunflower oil only during the challenge.

Test group.

After the challenge with the test item at a concentration of 50% (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 25% (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 50% (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 25% (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.