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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
The study was performed before implementation of the LLNA method (2010)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 11 October 2006 to 20 April 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: the study was performed according to internationally recognised guidelines and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was done in 2007and thus, before the implementation of the OECD TG for LLNA method (2010).

Test material

Constituent 1

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L’Arbresle, France
- Age at study initiation: on day 1, the animals of the main test were 1-2 months old
- Weight at study initiation (mean value on day 1): 369 and 392 g in control and treated males respectively, 384 and 398 g in control and treated females respectively
- Housing: individually in polycarbonate cages with stainless steel lid (48 cm x 27 cm x 20 cm)
- Diet: free access to 106 pelleted diet (SAFE, Villemoisson, Epinay-sur-Orge, France)
- Water: drinking water filtered by a FG Millipore membrane (0.22 micron), ad libitum
- Acclimation period: at least 5 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature:22 ± 2°C
- Humidity: 30 to 70%
- Air changes: approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12hrs dark / 12hrs light

IN-LIFE DATES: from 27 October 2006 (beginning of the preliminary test) to 20 November 2006 (sacrifice of the animals)

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
Preliminary test:
- By intradermal route: 0.05%, 0.1%, 0.5%, 1% and 2.5% (w/w)
- By cutaneous route: 5% and 10% (w/w)

Main test:
- Induction phase by intradermal route: 0.5% (w/w)
- Induction phase by cutaneous route: 10% (w/w)
- Challenge phase: 5% (w/w)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
Preliminary test:
- By intradermal route: 0.05%, 0.1%, 0.5%, 1% and 2.5% (w/w)
- By cutaneous route: 5% and 10% (w/w)

Main test:
- Induction phase by intradermal route: 0.5% (w/w)
- Induction phase by cutaneous route: 10% (w/w)
- Challenge phase: 5% (w/w)
No. of animals per dose:
- preliminary test: six animals (three males and three females)
- control group: ten animals (five males and five females)
- treated group: 20 animals (ten males and ten females)
Details on study design:
RANGE FINDING TESTS:
A preliminary test was conducted in order to determine the concentrations to be tested in the main study.
By intradermal route (tested concentrations: 0.05%, 0.1%, 0.5%, 1% and 2.5% (w/w)):
- intradermal injections of the dosage form preparations (0.1 mL) were performed in the interscapular region,
- local reactions were evaluated approximately 24, 48 hours and 6 days after the injections.
By cutaneous route:
- Under the conditions of the induction phase (tested concentrations: 5% and 10% (w/w)):
• a filter paper (approximately 8 cm2) was fully-loaded with a dosage form preparation and was then applied to the clipped area of the skin. The filter paper was held in place by means of an occlusive dressing for 48 hours,
• cutaneous reactions were evaluated 24 and 48 hours after removal of the dressing.
- Under the conditions of the challenge phase (tested concentrations: 5% and 10% (w/w)):
• the filter paper of a chamber (Finn Chamber®) was fully-loaded with a dosage form preparation. The chamber was then applied to the clipped area of the skin (one concentration per flank). The chamber was held in place by means of an occlusive dressing for 24 hours,
• cutaneous reactions were evaluated 24 and 48 hours after removal of the dressings.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (one intradermal and one cutaneous)
- type of epicutaneous induction: occlusive
- SLS application: no, as the test item was shown to be irritant during the preliminary test
- Exposure period: 48 hours (cutaneous induction)
- Test groups:
> injections with 50% (v/v) FCA (Freund complexe adjuvant) in 0.9% NaCl, or test item at 0.5% (w/w) in corn oil, or test item at 0.5% (w/w) in the mixture FCA/0.9% NaCl (50/50, w/w)
> cutaneous application: test item at the concentration of 10% (w/w) in corn oil
- Control group:
> injections with 50% (v/v) FCA in 0.9% NaCl, or vehicle (corn oil), or vehicle at 50% (w/v) in a mixture FCA/0.9% NaCl (50/50, v/v)
> cutaneous application: vehicle alone
- Site: the interscapular region of the animals
- Frequency of applications: not applicable
- Duration: 8 days (total duration of induction period)
- Concentrations: 0.5% (w/w) in corn oil (intradermal), 10% w/w in corn oil (epidermal)


B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: day 22
- Exposure period: 24 hours
- Test groups: the filter paper of a chamber (Finn Chamber®) was fully-loaded with the test item at the concentration of 5% (w/w) in corn oil and was then applied to a shaved area of the skin of the posterior right flank of all animals. The vehicle was applied under the same experimental conditions to the skin of the posterior left flank.
- Control group: the filter paper of a chamber (Finn Chamber®) was fully-loaded with the test item at the concentration of 5% (w/w) in corn oil and was then applied to a shaved area of the skin of the posterior right flank of all animals. The vehicle was applied under the same experimental conditions to the skin of the posterior left flank.
- Site: posterior right flank (test item) or posterior left flank (vehicle)
- Concentrations: 5% (w/w) in corn oil
- Evaluation: 24 and 48 hours after patch removal



OTHER: cutaneous reactions were evaluated according to the following scale:
- no visible change: 0
- discrete or patchy erythema: 1
- moderate and confluent erythema: 2
- intense erythema: 3
Any observed edema was recorded.
Any other lesions were noted.
Challenge controls:
Vehicle controls on the same animals (left flanks) + Control group (no contact with test substance during induction phase)
Positive control substance(s):
yes
Remarks:
mercaptobenzothiazole (CAS No 149-30-4)

Results and discussion

Positive control results:
Mercaptobenzothiazole was not included in the study but is regularly tested by the laboratory under the same conditions.
Based on the findings, the sensitivity of the guinea-pigs strain from the same source is considered satisfactory.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Negative control group in the control of the sensitivity of guinea pigs to a reference item (Mercaptobenzothiazole)
Dose level:
right flank (rmercaptobenzothiazole at the concentration of 20% (w/w))
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Negative control group in the control of the sensitivity of guinea pigs to a reference item (Mercaptobenzothiazole)
Dose level:
left flank (vehicle)
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
right flank (mercaptobenzotiazole at the concentration of 20% (w/w))
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
5 animals with erythema score 2 to 3, and showing edema and dryness of the skin. For the 5 remaining animals, the reading was masked by an important dryness of the skin.
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
left flank (vehicle)
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
right flank (rmercaptobenzathiazole at the concentration of 20% (w/w))
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
readness (10 animals, score 1 to 3), edema (in 2 anaimls), dryness of the skin ( animals)
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
left flank (vehicle)
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
other: Negative control group in the control of the sensitivity of guinea pigs to a reference item (Mercaptobenzothiazole)
Dose level:
right flank (Mercaptobenzothiazole at the concentration of 20% (w/w))
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
other: Negative control group in the control of the sensitivity of guinea pigs to a reference item (Mercaptobenzothiazole)
Dose level:
0 (Left flank, vehicle)
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle alone (left flank)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
5% (w/w) (right flank)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
velicle alone (left flank)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
5% (w/w) (right flank)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
dryness of the skin in 1 animal
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle alone (left flank)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
5% (w/w) (right flank)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
vehicle alone (left flank)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
5% (w/w) (right flank)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
dryness of the skin in 5 animals
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

Clinical examinations: no deaths and no clinical signs were noted during the study.

Body weight: the body weight change of the treated animals was similar to that of controls.

On removal of the dressing, no residual test item was observed.

No cutaneous reactions were observed in the animals of the control group.

In the treated group, no cutaneous reactions were noted on the left flank (vehicle application) of the animals. On the right flank (test item application), only a dryness of the skin was observed in 1/20 and 5/20 animals at the 24 and 48-hour readings, respectively.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Neodymium tris (di-2-ethylhexylphosphate) is not considered to be a skin sensitiser.
Executive summary:

In a dermal sensitization study using the Guinea Pig Maximisation Test method according to OECD guideline 406, EC B.6, US/EPA/OPPTS 870.2600 and GLP (CIT report No. 32371 TSG, 2007), scored as validity 1 according to Klimisch criteria, neodymium tris (di-2-ethylhexylphosphate) was administered to 1 to 2 months old Hartley Guinea pigs (5 controls and 10 treated/sex in the main test).

 

An induction treatment was carried out as follow:

On Day 1, 3 pairs of intradermal injections were performed in the interscapular region of all animals:

- Freund’s Complete Adjuvent (FCA) diluted to 50 % (v/v) with 0.9% NaCl (both groups),

- Test substance at the concentration of 0.5% (w/w) in corn oil (treated group) or vehicle alone (control group),

- Test substance at the concentration of 0.5% (w/w) in corn oil in a mixture of FCA/0.9 % NaCl (50/50, w/w) (treated group) or vehicle at the concentration of 50 % (w/v) in a mixture of FCA/0.9% NaCl (50/50, v/v) (control group).

On Day 8, the animals of the treated group received a topical application of the test item at the concentration of 10% (w/w) in corn oil to the same test site, which was then covered by an occlusive dressing for 48 hours. The animals of the control group received an application of the vehicle under the same experimental conditions.

 

On Day 22, all animals of both groups were challenged by a cutaneous application of the test item at the concentration of 5% (w/w) in corn oil to the right flank. The test item was maintained under an occlusive dressing for 24 hours. The vehicle was applied to the left flank under the same experimental conditions.

Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing, by grading for erythema and oedema.

 

No deaths and no clinical signs were noted during the study.

After the challenge application of the test item, no cutaneous reactions were observed in the animals of the control group.

In the treated group, no cutaneous reactions were noted on the left flank (vehicle application) of the animals.

On the right flank (test item application), only a dryness of the skin was observed in 1/20 and 5/20 animals at the 24 and 48-hour readings, respectively.

 

From the results obtained, neodymium tris (di-2-ethylhexylphosphate) is not classified as skin sensitizer according to Annex VI of the Directive 67/548/CEE and according to EU Regulation 1272/2008 (CLP).

 

This skin sensitisation study is classified as acceptable. It does satisfy the guideline requirement for a skin sensitisation study (OECD, EU, US) in the guinea pigs.