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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
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:
Acceptable guinea pig maximisation test that followed sound scientific principles

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): NExBTL Biodiesel
- Description: Clear colourless liquid
- Date received: 04 July 2006

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.