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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation, other
Remarks:
in vivo study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Groupe Interministeriel des Produits Chimiques, 15 February 2007
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Guinea Pig Maximation Test (GPMT) (Magnusson & Kligman, J Invest Dermatol. 1969;52:268-276) has been successfully used for predicting human skin sensitizers for more than 45 years. As a sensitive and reliable method it is considered the golden standard of sensitization assessment methods (Modjtahedi et al., Cutan Ocul Toxicol. 2011;30:129-137). Murine Local Lymph Node Assay (LLNA) has gained popularity as a new refinement method, and it is the recommended sensitization assessment method for REACH. However, the predictive ability of LLNA has been questioned due to the tendency for false positive results. The performance of LLNA and GPMT was compared in two studies by assessing the skin sensitizing properties of several chemically diverse organic compounds (Kreiling et al., Food Chem Toxicol. 2008;46:1896-1904; Ball et al., Regul Toxicol Pharmacol. 2011;60:389-400). The studies consistently indicated that LLNA overestimates the sensitizing potential of chemicals resulting in false positive sensitization classifications. Also endogenous compounds of human body and well-known non-sensitizing constituents of food and cosmetics were positive in LLNA. Modjtahedi et al. (2011) also questioned the predictive ability of LLNA as follows: “We note the current enthusiasm for the easier-to-perform LLNA but we suspect that in cases where the LLNA suggests that a chemical is a potent allergen, the guinea pig assays will prove productive for risk assessment.”
In accordance with these findings the outcome of LLNA suggested that NExBTL renewable diesel is a potential skin sensitizer although chemical analyses did not identify known sensitizers or other compounds that would explain the positive responses. Moreover, LLNA falsely identified two well-known non-sensitizers (kerosene and n-octadecane) as weakly positive. Therefore, the result of LLNA was not considered reliable. In contrast, the result of GPMT was clearly negative, which demonstrates that NExBTL is not a skin sensitizer.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): NExBTL renewable diesel
- Phycher internal code: PH-07/0460
- Description: colourless liquid
- Date received: 22 February 2008

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, F-69592 L'Arbresle, France
- Age at study initiation: approx. 4 wk
- Weight at study initiation: 230 - 278 g
- Housing: group housed
- Diet (e.g. ad libitum): yes (SDS C17.50 guinea pig breeder diet)
- Water (e.g. ad libitum): yes (tap water)
- Acclimation period: 5 d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24
- Humidity (%): 45-67
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES
Preliminary studies: 3-4 March 2008
Main study induction: 11 March 2008
Main study challenge: 31 March 2008

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
other: intradermal and epicutaneous (occlusive)
Vehicle:
other: olive oil (induction), liquid paraffin (challenge)
Concentration / amount:
INDUCTION
- intradermal injection (day 0): 25% in olive oil
- epicutaneous application (day 7); 100% (undiluted)

CHALLENGE
- epicutaneous application (day 20); 12.5% and 25% in liquid paraffin
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: olive oil (induction), liquid paraffin (challenge)
Concentration / amount:
INDUCTION
- intradermal injection (day 0): 25% in olive oil
- epicutaneous application (day 7); 100% (undiluted)

CHALLENGE
- epicutaneous application (day 20); 12.5% and 25% in liquid paraffin
No. of animals per dose:
Control: 10
Test: 20
Details on study design:
RANGE FINDING TESTS:
MAXIMAL NON-NECROTISING CONCENTRATION (MNNC)
- Objective: to determine concentration of test substance that does not cause severe necrosis after intradermal injection
- No. animals: 2
- Test site: scapular region
- Route: intradermal injection
- Concentration: 100%, 50%, 25%, 12.5%, 6.25% and 3.125% in olive oil
- Evaluation: sites assessed at 24 hr and 48 hr post-injection

PRE-MAXIMAL NON-IRRITANT CONCENTRATION (Pre-MNIC)
- Objective: to assess the irritancy of the test substance toward guinea pig skin, and determine if sodium lauryl sulphate should be used during topical induction phase
- No. animals: 2
- Test site: scapular region
- Route: epicutaneous, under occlusion (see below)
- Concentration: 50%, 25% and 12.5% in liquid paraffin
- Evaluation: sites assessed 24 hr post-application

MAXIMAL NON-IRRITANT CONCENTRATION (MNIC)
- Objective: to determine the maximum concentration of test substance that would be tolerated during topical challenge
- No. animals: 3
- Test site: dorso-lumbar
- Route: epicutaneous, under occlusion (see below)
- Concentration: 25%, 12.5%, 6.25% and 3.125% in liquid paraffin
- Evaluation: sites assessed 24 hr post-application

MAIN STUDY
A. INDUCTION EXPOSURE - INTRADERMAL INJECTION
- No. treatments: 1 injection, scapular region, day 0
- Exposure regime: animals received three (3) pairs of intradermal injections (0.1 ml per injection)
- CONTROL GROUP:
- 2 x Freund's Complete Adjuvant/isotonic sodium chloride, 1:1
- 2 x olive oil
- 2 x Freund's Complete Adjuvant/olive oil, 1:1
- TEST GROUP:
- 2 x Freund's Complete Adjuvant/isotonic sodium chloride, 1:1
- 2 x test substance, 25% in olive oil
- 2 x Freund's Complete Adjuvant/test substance, 1:1

B. INDUCTION EXPOSURE - EPICUTANEOUS OCCLUSIVE APPLICATION
- General: fur clipped from scapular region and 10% sodium lauryl sulphate in vaseline applied on day 6
- No. treatments: 1 epicutaneous application, scapular region, day 7
- Exposure regime: single occlusive application (0.5 ml) to region previously subject to i.d. injection
- CONTROL GROUP:
- 100% liquid paraffin
- TEST GROUP:
- 100% test substance
- Exposure period: occlusive wraps removed after 48 hr

C. CHALLENGE EXPOSURE - EPICUTANEOUS OCCLUSIVE APPLICATION
- No. treatments: 1 epicutaneous application, dorso-lumbar region, day 20
- Exposure regime: single occlusive application (0.5 ml)
- CONTROL AND TEST GROUPS:
- 12.5% and 25% test substance in liquid paraffin
- Evaluation (hr after challenge): 24 hr, 48 hr and 72 hr

D. OCCLUSIVE DRESSINGS
- Induction Phase: gauze (Lohmann and Rauscher) held in place with Micropore tape (3M) under Blenderm (3M)
- Challenge Phase: Hayes HA1 chamber, 8mm x 8mm

Challenge controls:
Challenge controls received 0.5 ml test substance, diluted in liquid paraffin (12.5%; 25%; as above)
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde (aHCA), CAS No 101-86-0

Results and discussion

Positive control results:
Clearly positive results obtained with positive control substance:
- 100% sensitisation with 50% aHCA at 24 and 48 hr post-challenge
- 50-100% sensitisation with 25% aHCA at 24 and 48 hr post-challenge

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
12.5% in liquid paraffin
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 12.5% in liquid paraffin. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
12.5% in liquid paraffin
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 12.5% in liquid paraffin. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
12.5% in liquid paraffin
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 12.5% in liquid paraffin. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25% in liquid paraffin
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25% in liquid paraffin. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25% in liquid paraffin
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25% in liquid paraffin. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
25% in liquid paraffin
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 25% in liquid paraffin. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
12.5% in liquid paraffin
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 12.5% in liquid paraffin. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
12.5% in liquid paraffin
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 12.5% in liquid paraffin. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
12.5% in liquid paraffin
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 12.5% in liquid paraffin. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25% in liquid paraffin
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
erythema grade 1 in 2 animals; erythema grade 2 in 1 animal
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25% in liquid paraffin. No with. + reactions: 3.0. Total no. in groups: 20.0. Clinical observations: erythema grade 1 in 2 animals; erythema grade 2 in 1 animal.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25% in liquid paraffin
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
erythema grade 1 in 1 animal
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25% in liquid paraffin. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: erythema grade 1 in 1 animal.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
25% in liquid paraffin
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 25% in liquid paraffin. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.

Any other information on results incl. tables

PRELIMINARY STUDIES

MNNC determination:

- slight necrosis following intradermal injection of 100% or 50% test substance in olive oil

- no necrosis at 25% or lower

- 25% was the MNNC and used for intradermal induction in the main test

Pre-MNIC determination:

- slight erythema present 24 hr after exposure to 100% or 50% test substance in liquid paraffin

- no erythema at 25% or lower

- 25% was the Pre-MNIC and used for epicutaneous induction

- 25% selected as the starting point for determination of the MNIC

MNIC determination

- no dermal reactions present at 24 hr

- 25% test substance in liquid paraffin was the MNIC

- 25% and 12.5% were selected as the challenge concentrations

MAIN STUDY

Induction phase:

- Control group

- no cutaneous reactions present after intradermal or epicutaneous treatment

- Test group

- no cutaneous reactions recorded after intradermal induction

- dryness (4/20 animals) and eschar (9/20 animals) present 24 hr after epicutaneous induction.

 

Challenge phase:

- Control group

- no cutaneous reactions at any time point (24, 48 or 72 hr)

Test group:

- slight or moderate erythema in 3/20 animals at 24 hr post-challenge with 25% test substance

- slight erythema in 1/20 animals at 48 hr post-challenge with 25% test substance

- no dermal responses in any animal 72 hr following challenge with 25% test substance

- no dermal responses in any animal at 24 hr, 48 hr or 72 hr after challenge with 12.5% test substance

WEIGHT GAIN

- negative control animals gained 159.5 g (SD = 30.7) over the course of the study

- test animals gained 150.6 g (SD = 23.4 ) over the same period

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Not a dermal sensitiser.
Executive summary:

The dermal sensitisation potential of NExBTL renewable diesel was assessed in a GLP-compliant guideline guinea pig maximisation test (skin sensitisation, method B6 of directive 96/54/EC) using 10 control animals and 20 test animals. Based on results of preliminary testing, concentrations of 25% in olive oil (intradermal injection) and 100% (epicutaneous, under occlusion) were administered during the induction phase (study days 0 and 7, respectively) with 12.5% and 25% in liquid paraffin (epicutaneous, under occlusion) used for challenge on study day 20. Dermal responses (erythema, oedema) were recorded at 24, 48 and 72 hr post-challenge. Alpha-hexylcinnamaldehyde (aHCA; n=10 animals) was used as positive control substance. No dermal reactions were present in the negative controls following challenge with NExBTL renewable diesel, however slight or moderate erythema was present in 3/20 animals 24 hr after challenge with 25%, with slight erythema remaining in 1/20 animals at the 48 hr time point. These reactions were fully resolved by 72 hr. No erythema or oedema was in any test animal challenged with 12.5% test substance. A satisfactory response was obtained with the positive control group (100% sensitisation at 50% aHCA; 50-100% with 25%). Both the control (+159.5 g; SD = 30.7) and test groups (150.6 g; SD = 23.4) gained weight over the course of the study. The results demonstrate that NExBTL renewable diesel is not a skin sensitiser.