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Diss Factsheets

Administrative data

Description of key information

Multiple skin sensitization studies are available for the substance. This includes two LLNA, 2 Guinea Pig Maximization tests, a Buehler assay and a human patch test study. All of these tests provided negative results except one of the Guinea Pig Maximization tests which was positive.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Remarks:
other: Human volunteer study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Report of apparently well conducted human volunteer trial with detail of volunteer panel and reactions to treatment. Test material said to be directly comparable to pinene oliogomers (and reported characteristics support this). Study findings are supported by a separate (incompletely reported) human repeat-insult patch test of the same test substance in which 50 male volunteers apparently showed no skin irritation or sensitisation reactions.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Closed patch applied to skin of human volunteers for 24h: after recording of irritation, challenge by repeat patching 10-14 days later
GLP compliance:
not specified
Type of study:
other: Investigation of primary irritation and contact sensitisation using the Closed Patch procedure
Species:
human
Sex:
male/female
Details on test animals and environmental conditions:
42 adult males, 18-57 years old, 11 with declared allergies. 15 adult females, 18-57 years old, 4 with declared allergies.
Reading:
other: primary irritation evaluation after 1st patch removal
Group:
test chemical
Dose level:
Undiluted test substance
No. with + reactions:
0
Total no. in group:
57
Clinical observations:
No skin reactions observed
Remarks on result:
other: Reading: other: primary irritation evaluation after 1st patch removal. Group: test group. Dose level: Undiluted test substance. No with. + reactions: 0.0. Total no. in groups: 57.0. Clinical observations: No skin reactions observed.
Reading:
other: sensitisation evaluation after 2nd patch removal
Group:
test chemical
Dose level:
undiluted test substance
No. with + reactions:
0
Total no. in group:
53
Clinical observations:
No skin reactions observed
Remarks on result:
other: Reading: other: sensitisation evaluation after 2nd patch removal. Group: test group. Dose level: undiluted test substance. No with. + reactions: 0.0. Total no. in groups: 53.0. Clinical observations: No skin reactions observed.

No skin reactions indicative of primary irritation or contact sensitisation were observed in human volunteers.

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under the conditions of this study, no evidence of human contact sensitisation was obtained.
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP, using multiple, topical induction treatments to optimise the possibilities for skin penetration
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
other: Enhanced Buehler test: 9 topical inductions
Justification for non-LLNA method:
Test conducted prior to creation of LLNA guideline (OECD 429, adopted 2002)
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: David Hall Limited, Darley Oaks, Newchurch, Burton-on-Trent, Staffordshire
- Age at study initiation: < 1 year old
- Weight at study initiation: ranged from 375 - 420 g
- Housing: Test animals were housed in aluminium cages (48 x 61 x 25 cm) with a grid floor. Dose ranging animals were housed 2 per cage and the main study animals were housed 5 per cage.
- Diet: Guinea Pig Diet FD1 (supplied by Special Diet Services Ltd, 1 Stepfield Witham, Essex, CM8 3AD) was available ad libitum
- Water: domestic mains quality drinking water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 20
- Humidity (%): 49
- Air changes (per hr): 15 - 20
- Photoperiod: 12 hours light / 12 hours dark

Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Remarks:
for the main study, the test material was used as supplied. For the Dose ranging study, maize oil was used as a vehicle.
Concentration / amount:
For the dose range finding study, maize oil was used a vehicle for the test substance. For the main test, the test substance was used as supplied.
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Remarks:
for the main study, the test material was used as supplied. For the Dose ranging study, maize oil was used as a vehicle.
Concentration / amount:
For the dose range finding study, maize oil was used a vehicle for the test substance. For the main test, the test substance was used as supplied.
No. of animals per dose:
34 young adult Dunkin-Hartley female, nulliparous and non-pregnant, albino Guinea pigs were used in this study (dose rangefinder 4 animals, then 20 test animals 10 animals negative controls).
Details on study design:
RANGE FINDING TESTS:
A total of 4 animals and 8 dose levels were used for the dose range finding study. The doses chosen for the main study were based on the results of the dose ranging study and took account of any irritation potential of the test material observed during the test.

The induction and challenge phases were performed by topical application of the test material. Prior to the first dose, hair was clipped from both flanks of each animal (to give an approximate testing site area of 4 cm x 6 cm). The next day animals were treated with the test material by topical application to the flanks under Webril patches (2.5 cm x 2.5 cm). Aluminium foil was used to cover each patch and Blenderm occlusive tape and elastic bandages were wrapped around the torso of the animal to secure the patch. Patches were removed after 6 h. Any residual test material on test sites were wiped using sterile distilled water. This procedure was carried out for 3 consecutive days.

MAIN STUDY
A. INDUCTION EXPOSURE

Prior to the first application each week, hair was clipped from the left flank of animals (to give a test site area of approximately 5 cm x 5 cm). The following day each animal received topical application of the test material to the left flank under a Webril patch (2.5 cm x 2.5 cm). Aluminium foil was used to cover each patch and Blenderm occlusive tape and elastic bandage were wrapped around the torso of the animal to secure the patch. Patches were removed after 6 h. Any residual test material on test sites was wiped using sterile distilled water. This procedure was conducted on 3 consecutive days for each of 3 weeks.

Examination of test sites occurred after 18 h post patch removal

B. CHALLENGE EXPOSURE

13 days after the final induction application, hair was clipped from the right flank of animals to give a test site area of 6 cm x 4 cm. Two sets of test patches for challenge were prepared, to investigate possible differences between UV-irradiated and non-irradiated test substance:
[1] At 24 hours prior to dosing, test material (0.5 ml) was applied to 30 Webril patches (2.5 cm x 2.5 cm). These patches were irradiated with UV radiation for 2 hours (UVA and UVB intensity measured using UVX-31 and UVX-36 sensors). These UV-exposed patches were then stored in the dark until use.
[2] 22 h later, 30 Webril patches (2.5 cm x 2.5 cm) were treated with 0.5 ml test material immediately prior to application.
Patches from both sets ([1] and [2] were then applied to the animals and covered with aluminium foil plus Blenderm occlusive tape; elastic bandages were wrapped around the torsos of the animals to secure the patches. Patches were removed after 6 h. Any residual test material on test sites was wiped using sterile distilled water.

Examination of test sites occurred after 24 h and 48 h post patch removal. Bodyweights of each individual animal were also recorded prior to the induction procedure and after the final test site observation at challenge.
Positive control substance(s):
yes
Remarks:
Hexylcinnamaldehyde (HCA: 75% in acetone/PEG 400, 70:30 v/v) was used in a separate study confirming test method sensitivity (positive control check) .
Positive control results:
80% of the positive control group (treated with HCA) showed a positive response. No positive responses were noted in the concurrent control group.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100% (non-irradiated)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No clinical signs were seen
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100% (non-irradiated). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No clinical signs were seen.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100% (non-irradiated)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No clinical signs were seen
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100% (non-irradiated). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No clinical signs were seen.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100% (irradiated)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No clinical signs were seen
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100% (irradiated). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No clinical signs were seen.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100% (irradiated)
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
Single test animal showed score 1 at 48h post challenge. No clinical signs were seen
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100% (irradiated). No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: Single test animal showed score 1 at 48h post challenge. No clinical signs were seen.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100% (non-irradiated)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No clinical signs were seen
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 100% (non-irradiated). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No clinical signs were seen.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100% (irradiated)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No clinical signs were seen
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 100% (irradiated). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No clinical signs were seen.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
75% HCA
No. with + reactions:
16
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, the test substance showed no skin sensitising activity in Guinea pigs. This was true for both types of challange treatment (application of undiluted test material, either as supplied or UV-irradiated).
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Test conducted prior to creation of LLNA guideline (OECD 429, adopted 2002)
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire, England
- Age at study initiation: 6-7 weeks
- Weight at study initiation: ranged from 288 - 325 g
- Housing: Test animals were housed in groups of 5 in suspended metal cages with wire mesh floors.
- Diet: Vitamin C enriched guinea-pig diet FDI ad libitum. Hay was given weekly
- Water: ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod: 12 hours light / 12 hours dark
Route:
intradermal and epicutaneous
Vehicle:
water
Remarks:
(distilled)
Concentration / amount:
Preliminary study:
- Induction (intradermal): 0.1% v/v in water
- Induction (topical): 40% v/v in distilled water
- Challenge (topical): 40 and 20% v/v in distilled water

Definitive study
- Induction (intradermal): [1] Freunds complete adjuvant diluted with equal volum of water, [2] test substance 0.1% v/v in water, [3] test substance 0.1% v/v in a 50/50 mixture of Freunds complete adjuvant and water.
- Induction (topical): test substance 40% v/v in distilled water
For Induction, control animals were treated similarly to test animals except that test substance was omitted.
- Challenge (topical): 40% and 20% v/v in distilled water (for both control and test animals)
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
(distilled)
Concentration / amount:
Preliminary study:
- Induction (intradermal): 0.1% v/v in water
- Induction (topical): 40% v/v in distilled water
- Challenge (topical): 40 and 20% v/v in distilled water

Definitive study
- Induction (intradermal): [1] Freunds complete adjuvant diluted with equal volum of water, [2] test substance 0.1% v/v in water, [3] test substance 0.1% v/v in a 50/50 mixture of Freunds complete adjuvant and water.
- Induction (topical): test substance 40% v/v in distilled water
For Induction, control animals were treated similarly to test animals except that test substance was omitted.
- Challenge (topical): 40% and 20% v/v in distilled water (for both control and test animals)
No. of animals per dose:
- Control animals: 5
- Test animals (receiving test material): 10
Details on study design:
RANGE FINDING TESTS:

A preliminary range-finding study was conducted to identify a concentration of test substance that would produce sufficient irritation for the induction phase of the main study and a maximal, non-irritant concentration for topical administration of the test substance in the challenge phase.

The preliminary investigation determined the following concentrations of Nu-Film-17 (Pinolene) should be used in the main study:
- Induction (intradermal injection): 0.1% v/v in water (localised necrosis seen at higher concentrations - only slight at 0.25%)
- Induction (topical application): 40% v/v in distilled water
- Challenge (topical application): 40 and 20% v/v in distilled water

MAIN STUDY
A. INDUCTION EXPOSURE

Induction (intradermal injection): An area (40 x 60 mm) of the dorsal skin on the interscapular region of the guinea-pig was clipped free of hair. Three pairs of intradermal injection were made within the clipped area. Test animals received the following injections:
1) Freund's complete adjuvant was diluted with an equal volume of water.
2) test substance, 0.1% v/v in water
3) test substance, 0.1% v/v in a 50:50 mixture of Freund's complete adjuvant
[Volumes of 0.1 ml were injected into both left and right injection sites]

Induction (topical application): A week after induction by intradermal injection, the same 40 x 60 mm interscapular region was clipped free of hair. A 20 x 40 mm patch of Whatman No. 3 paper was saturated with 0.4 ml of test substance, 40% v/v in distilled water. This patch was placed onto the clipped region of the test animal and covered with impermeable plastic adhesive tape (50 mm width 'Blenderm'). This was further secured by elastic adhesive bandage (50 mm width 'Elastoplast') wrapped around the torso of the animal and fixed with 'Sleek' impervious plastic adhesive tape. The dressing was left in place for 48 hours.

Induction of control animals: Control animals were treated in a similar fashion to the test animal except that the test substance was omitted from the intradermal injections and topical application.

B. CHALLENGE EXPOSURE (for both test and control animals)

Two weeks after the topical induction, control and test animals were challenged by topical application of test substance, 40 and 20% v/v in distilled water. An area on the left flank of each guinea-pig was clipped free of hair and a 20 x 20 mm patch of Whatman No.3 was saturated with 0.2 ml of test substance, 40% v/v in distilled water and applied to the anterior site on the flank. A similar patch prepared with 20% v/v test substance in distilled water was applied to the posterior site. Patches were sealed under strips of 'Blenderm' covered by 'Elastoplast' wrapped around the trunk and secured with 'Sleek' for 24 hours.
Challenge controls:
See above ('Details on study design') for challenge controls.
Positive control substance(s):
yes
Remarks:
Hexylcinnamic aldehyde was used in a separate study confirming test method sensitivity (positive control check) .
Positive control results:
The sensitivity and reliability of the test system was assessed in separate studies with hexyl cinnamic aldehyde (HCA). In 3 control studies conducted between 08 Dec 1992 and 11 April 1994, tests conducted with HCA gave acceptable positive responses.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
40% (Anterior site)
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
5 reactors showed scores greater than any negative control score
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 40% (Anterior site). No with. + reactions: 8.0. Total no. in groups: 10.0. Clinical observations: 5 reactors showed scores greater than any negative control score.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
20% (Posterior site)
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
No reactor showed score greater than any negative control score
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 20% (Posterior site). No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: No reactor showed score greater than any negative control score.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
40% (Anterior site)
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
2 reactors showed scores greater than any negative control score
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 40% (Anterior site). No with. + reactions: 6.0. Total no. in groups: 10.0. Clinical observations: 2 reactors showed scores greater than any negative control score.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
20% (Posterior site)
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
No reactor showed score greater than any negative control score
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 20% (Posterior site). No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: No reactor showed score greater than any negative control score.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
40% (Anterior site)
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
5 reactors showed scores greater than any negative control score
Remarks on result:
other: see Remark
Remarks:
Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 40% (Anterior site). No with. + reactions: 6.0. Total no. in groups: 10.0. Clinical observations: 5 reactors showed scores greater than any negative control score.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
20% (Posterior site)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 20% (Posterior site). No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
40% (Anterior site)
No. with + reactions:
1
Total no. in group:
5
Clinical observations:
A single animal showed localised reaction (across part of challenge site), score 1
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 40% (Anterior site). No with. + reactions: 1.0. Total no. in groups: 5.0. Clinical observations: A single animal showed localised reaction (across part of challenge site), score 1.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
20% (Posterior site)
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 20% (Posterior site). No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
40% (Anterior site)
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 40% (Anterior site). No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
20% (Posterior site)
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 20% (Posterior site). No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
40% (Anterior site)
No. with + reactions:
1
Total no. in group:
5
Clinical observations:
A single animal showed localised reaction (across part of challenge site), score 1
Remarks on result:
other: see Remark
Remarks:
Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 40% (Anterior site). No with. + reactions: 1.0. Total no. in groups: 5.0. Clinical observations: A single animal showed localised reaction (across part of challenge site), score 1.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
20% (Posterior site)
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 20% (Posterior site). No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Group:
positive control
Dose level:
10%; 50% and as supplied
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Under the conditions of this maximisation test, 50% of test animals showed skin reactions greater than the maximum reaction to challenge seen in any negative control animal (and 50% showed reactions persisting across the 24-48-72h observations). This leads to a conclusion that the test substance caused delayed contact sensitisation.
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Test conducted predates adoption of LLNA OECD test guideline
Species:
guinea pig
Strain:
Hartley
Remarks:
Hartley-Albino
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, MA
- Females (if applicable) nulliparous and non-pregnant: Not specified
- Microbiological status of animals, when known: Not specified
- Age at study initiation: Not specified
- Weight at study initiation: Males (328-406g) and females (323-368g)
- Housing: House individually during wrapping and 1-4 animals per cage at other times. Housed in suspended, wire bottom, stainless steel cages.
- Diet (e.g. ad libitum): PMI Feeds Inc. Guinea pig diet #5025 available ad libitum
- Water (e.g. ad libitum): Municipal water available ad libitum
- Acclimation period: 5 days
- Indication of any skin lesions: None noted

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±3°C
- Humidity (%): 30-80%
- Air changes (per hr): 10-12 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100% v/v
Day(s)/duration:
Day 7
Adequacy of induction:
not specified
Route:
intradermal
Vehicle:
water
Concentration / amount:
3% v/v
Day(s)/duration:
Day 0
Adequacy of induction:
not specified
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100% v/v
Day(s)/duration:
Day 21
Adequacy of challenge:
not specified
No. of animals per dose:
5 per sex per group
Details on study design:
RANGE FINDING TESTS: Not specified

MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal injections: The animals were treated on Day 0 by making three pairs of symmetrical intradermal injections on the upper back of each animal within a 4 x 6 cm exposure area running laterally across the shoulders. For the test animals (group 1), the first pair of injections (one on each side of the spinal column and approximately 3.5 cm apart), consisting of Freund's complete adjuvant diluted 50% v/v in saline, was made at the anterior edge of the exposure area. The second pair of injections, consisting of 3% v/v test substance in deionized water, was made approximately 0.5 cm behind the first pair. the third pair of injections, consisting of 50:50 mixyure of Freund's Complete adjuvant (diluted to 50% v/v in saline) and a solution of 3% v/v test substance in deionized water was made approximately 0.5 cm behind the second pair of injections. Group 2 control animals received the same injections with the vehicle substituted for the test substance in the second and third pairs of injections. All injections were within a 2 x 4 cm exposure area. A volume of 0.1 mL was administered at each site.

Topical applications: On Day 7, 0.5 mL of undiluted test substance was applied to the exposure area of each test group animal (Group 1) to cover the intradermal injection sites. A 5 cm round patch of filter paper was used to cover the dose site. The patch was then occluded with an adhesive masking tape and secured in place with an elastic adhesive wrap wound around the torso of the animal. Control group animals (Group 2) received 0.5 mL of vehicle and a 5 cm patch of filter paper was placed over the dose sites. The wrappings and patched were removed after 48 hours. Test sites 3 and 4 were observed for dermal irritation on day 10.

B. CHALLENGE EXPOSURE
On Day 21, a 5 x 5 cm area was clipped on both the left and right flanks of each test and control animal. For the challenge treatment, 0.5 mL of undilued test substance was applied topically to the right flank of each animal in a manner identical to the Day 7 treatment. A 5 cm round patch of filter paper was used to cover the dose site. A dry 5 cm round patch of filter paper was applied topically to the left flank of each animal. Patches were secured as above.

OTHER: On Day 22 (24 hours after challenge), the wrappings and patches were removed. On Day 23 (24 hours after unwrapping), the test sites were observed for skin reactions and again observed on Day 24 (48 hours after unwrapping). Observations were made of right and left flanks of each animal in Groups 1 and 2. After both induction treatments and the challenge exposure, average skin reaction scores were calculated.

Any Group 1 animals which exhibited scores greater than 0 for erythema with or without edema for the treated right flank after the challenge treatment were considered possibly sensitiszed. However, the skin reactions of the left flank treated with patch alone and the skin reactions of the naive controls were also evaluated.
Challenge controls:
Challenged with 100% v/v of test substance.
Positive control substance(s):
yes
Remarks:
1-chloro-2,4-dinitro-benzene (0.5% w/v solution of test substance in cottonseed oil with and without adjuvant (50:50 v/v) for intradermal injections; 2% w/w concentration of test substance in petrolatum for topical insult and challenge application.
Positive control results:
Since all 20 animals of the test group exhibited patchy to intense erythema after the challenge treatment, and only three of the 10 animals of the naive control group exhibited patchy to moderate erythema after the challenge treatment, the test substance is considered an extreme sensitizer and confirmed the sensitivity of guinea pigs to the positive control material.
Reading:
2nd reading
Hours after challenge:
72
Group:
other: Positive control; control group
Dose level:
2% v/v in petrolatum
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
None noted
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
other: Positive control; control group
Dose level:
2% v/v in petrolatum
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
None noted
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
positive control
Dose level:
2% w/w in petrolatum
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
None noted
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Dose level:
2% w/w in petrolatum
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
None noted
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
Vehicle
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None noted
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
Vehicle
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None noted
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
100% v/v
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None noted
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100% v/v
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None noted
Remarks on result:
no indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
Since none of the test animals exhibited scores greater than zero, the test substance was not considered a skins sensitizer.
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: Mouse; CBA/JCr
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley; Indianapolis IN.
- Females (if applicable) nulliparous and non-pregnant: Not specified
- Microbiological status of animals, when known: Not specified
- Age at study initiation: At least 6 weeks old
- Weight at study initiation: 17.1 to 20.6g
- Housing: Individually housed in polycarbonate cages with stainless steel covers
- Diet (e.g. ad libitum): PMI Feeds Inc. Formulab #5008 available ad libitum
- Water (e.g. ad libitum): Municipal water supplied ad libitum
- Acclimation period: At least 5 days
- Indication of any skin lesions: None noted

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10-12 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
100% (undiluted), 50 and 25% v/v in acetone/olive oil (4:1 v/v)
No. of animals per dose:
5 females per group
Details on study design:
MAIN STUDY
5 animals per group (3 groups, control and positive control) were treated with an appropriate volume of the test substance/vehicle. Each animal received 25 µL to the dorsum of each ear. The animals were treated once daily for 3 days. After a 2 day rest period, all animals were injected with tritiated methyl-thymidine in the tail vein. Five hours later, the animals were sacrificed and the draining auricular lymph nodes removed and prepared for cell suspension and scintillation counting. A vehicle control group of 5 animals was treated in the same manner with vehicle instead of test substance or dilution. A positive control group of 5 females was also run one week later and treated with 25% alpha-hexylcinnamaldehyde in vehicle.


ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
1) Ratio of DPM relative to that recorded for control lymph nodes (SI) is derived for each test group based on the group mean DPM.
2) Positive response includes SI > 3 with consideration of dose response and where appropriate statistical significance.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of appropriate concentration applied directly to the dorsum of both ears.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not required
Positive control results:
The SI was 5.1 which was > 3 indicating a positive response and valid test
Parameter:
SI
Value:
12.2
Variability:
NA - Pooled method
Test group / Remarks:
Positive Control
Parameter:
SI
Value:
1.3
Variability:
NA - Pooled method
Test group / Remarks:
100% v/v
Parameter:
SI
Value:
0.9
Variability:
NA - Pooled approach
Test group / Remarks:
50% v/v
Parameter:
SI
Value:
0.9
Variability:
NA - Pooled approach
Test group / Remarks:
25% v/v
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION: Ratio of DPM relative to that recorded for control lymph nodes (SI index) is derived for each test group based on group mean DPM.

EC3 CALCULATION: Not possible to calculate (all SI values less than 3).

CLINICAL OBSERVATIONS: All animals appeared normal for the duration of the study.

BODY WEIGHTS: There was no effect on bodyweight gain during the study.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness): None noted.

Group DPM counts and SI values
















































Animal group



Test substance concentration



Average count per mouse



Group size



SI



Vehicle control



NA



3911



5



NA



Test group 1



25%



3488



5



0.9



Test group 2



50%



3491



5



0.9



Test group 3



100%



4991



5



1.3



Positive control



NA



47767



5



12.2


Interpretation of results:
GHS criteria not met
Conclusions:
The test substance produced a stimulation index of < 3 in all groups of test animals and is not therefore considered a skin sensitizer. The positive control substance showed the expected response demonstrating test validity.
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley; Indianapolis IN.
- Females (if applicable) nulliparous and non-pregnant: Not specified
- Microbiological status of animals, when known: Not specified
- Age at study initiation: At least 6 weeks old
- Weight at study initiation: 15 to 20.9g
- Housing: Individually housed in polycarbonate cages with stainless steel covers
- Diet (e.g. ad libitum): PMI Feeds Inc. Formulab #5008 available ad libitum
- Water (e.g. ad libitum): Municipal water supplied ad libitum
- Acclimation period: At least 5 days
- Indication of any skin lesions: None noted

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10-12 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
100% (undiluted), 50 and 25% v/v in acetone/olive oil (4:1 v/v)
No. of animals per dose:
5 females per group
Details on study design:
MAIN STUDY
5 animals per group (3 groups, control and positive control) were treated with an appropriate volume of the test substance/vehicle. Each animal received 25 µL to the dorsum of each ear. The animals were treated once daily for 3 days. After a 2 day rest period, all animals were injected with tritiated methyl-thymidine in the tail vein. Five hours later, the animals were sacrificed and the draining auricular lymph nodes removed and prepared for cell suspension and scintillation counting. A vehicle control group of 5 animals was treated in the same manner with vehicle instead of test substance or dilution. A positive control group of 5 females was also run one week later and treated with 25% alpha-hexylcinnamaldehyde in vehicle.


ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
1) Ratio of DPM relative to that recorded for control lymph nodes (SI) is derived for each test group based on the group mean DPM.
2) Positive response includes SI > 3 with consideration of dose response and where appropriate statistical significance.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of appropriate concentration applied directly to the dorsum of both ears.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not required
Positive control results:
The SI was 5.1 which was > 3 indicating a positive response and valid test
Key result
Parameter:
SI
Value:
5.1
Variability:
NA - Pooled method
Test group / Remarks:
Positive Control
Parameter:
SI
Value:
1.5
Variability:
NA - Pooled method
Test group / Remarks:
100% v/v
Parameter:
SI
Value:
1.4
Variability:
NA - Pooled approach
Test group / Remarks:
50% v/v
Parameter:
SI
Value:
1.2
Variability:
NA - Pooled approach
Test group / Remarks:
25% v/v
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION: Ratio of DPM relative to that recorded for control lymph nodes (SI index) is derived for each test group based on group mean DPM.

EC3 CALCULATION: Not possible to calculate (all SI values less than 3).

CLINICAL OBSERVATIONS: All animals appeared normal for the duration of the study.

BODY WEIGHTS: There was no effect on bodyweight gain during the study, except in one positive control animal that failed to gain weight.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness): None noted.

Group DPM counts and SI values
















































Animal group



Test substance concentration



Average count per mouse



Group size



SI



Vehicle control



NA



9406



5



NA



Test group 1



25%



11786



5



1.2



Test group 2



50%



13497



5



1.4



Test group 3



100%



14335



5



1.5



Positive control



NA



47950



5



5.1


Interpretation of results:
GHS criteria not met
Conclusions:
The test substance produced a stimulation index of < 3 in all groups of test animals and is not therefore considered a skin sensitizer. The positive control substance showed the expected response demonstrating test validity.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Guinea pig and mice skin sensitisation studies are available, plus a human skin sensitisation study: all are considered reliable. One Guinea pig study followed the maximisation test procedure, with intradermal injection and adjuvant to optimise immune system reactions: this gave a positive result (50% of test animals showing greater reaction to challenge than controls and the same proportion showing persistent reactions). The other Guinea pig study used multiple topical inductions (Enhanced Buehler method) and gave a clearly negative result. A further guinea pig maximization test also provided negative results. Two mice local lymph node assays are also available providing negative results. 



The human volunteer test found no evidence of skin sensitisation following a single topical induction and topical challenge: this result is supported by a separate study (not otherwise described in this dossier due to incomplete reporting) which employed 10 separate, 24h topical inductions followed 2 weeks later by one topical 48h challenge and found no skin reactions in 50 male volunteers.


Based on a weight-of-evidence approach examining the available animal and human data, the substance is not considered to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

No animal test of respiratory sensitisation has been identified. Falk AA et al. (Scand. J. Work Environ. Health 16: 372-8, 1990) reported that 5 of 8 human volunteers exposed to alpha-pinene by inhalation (2h exposures during light exercise) at 450 mg/cu.m reported irritation of the eyes, nose and throat: however no significant change in lung function was found after exposure, with no indication of bronchoconstriction.


Falk Filipsson, Bard and Karlsson (CICAD 5 – Limonene, WHO 1998) noted that 8 human subjects exposed by inhalation to d-limonene (2h at up to 450 mg/cu.m) reported no discomfort or irritation. Taken together with the skin sensitisation reported here, these data for close analogues of pinene oligomers do not provide sufficient evidence to designate pinene oligomers as a respiratory sensitiser.


 


 

Justification for classification or non-classification

The substance is not classified in accordance with EU CLP regulation (EC No. 1272/2008, as amended) based on a self-classification. 


The available skin sensitisation data available from one guinea pig maximisation test potentially indicates that at the top challenge dose (0.1% intradermal induction dose) of 40%, >/= 30% responded to challenge, however this number dropped to < 30% at the lower challenge dose of 20%. An additional test using a guinea pig enhanced Buehler method (more realistic conditions to that potentially possible for human exposure), showed that no skin sensitisation occurred (negative). There is also evidence from patch testing in humans, that the substance is not a skin sensitiser alongside negative results from two LLNAs and a further guinea pig maximization test.


Based on a weight-of-evidence approach examining the available animal and human data, the substance is not considered a skin sensitiser and classification under CLP is not warranted.