Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Four in vivo sensitisation studies were conducted to evaluate the sensitisation potential of alpha-pinene and beta-pinene oligomers; OECD 406 guinea pig maximisation assay (Richeux, 2007), two local lymph node assay (LLNA) studies, OECD 429 (Sanders, 2012a; Sanders, 2012b) and LLNA Brd-U, OECD 442B (Richeux, 2018). Three of the four studies resulted in a non-sensitising outcome, however, the LLNA Brd-U study (Richeux, 2018) concluded that under the experimental conditions, alpha-pinene and beta pinene oligomers resulted in a sensitising outcome. 

 

A thorough review of the available data from all four studies was conducted to understand why there was a conflicting outcome. In all three LLNA studies, different concentrations and different vehicles were used. Two LLNA studies reported difficulties in obtaining a suitable vehicle, acetone:olive oil 4:1 v/v was not considered suitable for dosing in either of the OECD 429 studies (Sanders 2012a; sanders, 2012b). The third LLNA (Richeux, 2018) only considered acetone:olive oil (4:1 v/v). Skin irritation in the form of increased ear puncture weights (>25%) was recorded in all three Local Lymph Node Assay studies. The highest concentration of 50% was used in OECD 442B study whereas the OECD 429 study (Sanders, 2012a) considered 50% to cause skin irritation based on an increase in ear thickness in the preliminary screening test and subsequently not used in the assay in accordance with the OECD 429 test guidance.  The second OECD 429 study (Sanders, 2012b) could not formulate a suitable dosing preparation at a concentration of 50% in any vehicle.

 

Based on the reported data, it is considered that the concentrations tested in the OECD 442B study (Richeux, 2018) elicited excessive skin irritation, illustrated by an increase in ear puncture weights, potentially leading to a false positive outcome. Pre-screen tests in the two other OECD 429 studies (Sanders 2012a; Sanders, 2012b) demonstrated that alpha-pinene and beta-pinene oligomers caused skin irritation at concentrations greater than 25%. This was corroborated in the guinea pig maximisation test, where skin irritation was recorded at concentrations greater than 25% in the preliminary irritation tests. The most appropriate concentration for the challenge exposure was 25%, the maximum non irritating concentration in the guinea pig (Richeux, 2007). This study can also be used as supporting evidence that alpha-pinene and beta-pinene oligomers are not expected to cause skin sensitisation.

 

Skin irritation and the vehicle choice influenced the outcome of these studies resulting in a false positive LLNA Brd-U outcome. The choice of concentrations used in the OECD 442B study (Richeux, 2018) are not compliant with the relevant OECD test guidelines for this study type (OECD 442B (OECD, 2018)) due to the increased ear puncture weights. It is considered that this study was conducted using concentrations which caused excessive skin irritation in a potentially inappropriate vehicle based on the evidence supplied in the available study reports. 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 February - 10 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA
Species:
mouse
Strain:
other: CBA/J (CBA/JRj)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elevage Janvier Labs, Le Genest-Saint-Isle, France.
- Age at study initiation: 8 weeks
- Weight at study initiation: g
- Housing: Animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood wood flakes.
- Diet: Teklad Global 16% Protein Rodent Diet (Envigo, 2016), ad libitum
- Water: Drinking water (tap water from public distribution system), ad libitum
- Acclimation period: at least5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: 10 changes/h
- Photoperiod: 12 h dark / 12 h light
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Preliminary test: 100 % and 50 %
Main test: 10, 25 and 50 %
No. of animals per dose:
Preliminary test: 2 animals
Main test: 4 females/dose
Details on study design:
PRELIMINARY TEST:
- The mice were treated by daily application of 25 μL of the test item undiluted (100 %) or at 50% in AOO to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were observed daily from day 1. Any signs of toxicity or excessive local irritation noted during this period were recorded. Ear thickness was recorded on Days 1, 3 and 6.
- Cutaneous reactions were noted at the concentration of 100 % but not at 50%. Therefore 50% was chosen as the highest concentration for the main study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT:
- Animals were randomly allocated to the groups.
- Name of test method: Local Lymph Node Assay (LLNA:BrdU)
- Criteria used to consider a positive response: Test item should be classified if at least one concentration of the test item results in a SI≥ 1.6, as sensitiser. Any test item failing to produce a SI> 1.6 was classified as a "non-sensitiser". However, the strength of the dose-response relationship, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result (i.e. SI value between 1.6 and 1.9) is declared positive.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of control and test item were applied to the dorsal surface of both ears on Days 1, 2 and 3. On Day 5, 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution was injected by intra-peritoneal route. On day 6 (end of the test), the animals were were sacrificed by pentobarbital sodium and the auricular lymph nodes were excised. Then, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by measurement of BrdU content in DNA of lymphocyte with ELISA kit.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No data
Positive control results:
Based on the EC1.6 of the current positive control, tested with HCA, the positive control is classified as sensitiser, as expected.
Parameter:
SI
Value:
1.34
Test group / Remarks:
Treated groups at 10%
Parameter:
SI
Value:
1.65
Test group / Remarks:
Treated group at 25%
Parameter:
SI
Value:
2.08
Test group / Remarks:
Treated group at 50%
Key result
Parameter:
other: EC1.6
Remarks:
%
Value:
22.58

Clinical observations and mortality

No mortality and no signs of systemic toxicity were noted in the test and control animals during the test.

Bodyweights

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Local irritation

Dryness of the skin was noted on Day 6 in all animals treated with test item at 25 and 50%.

An increase in ear weight (+3%, +27% +47.8%) was noted in animals treated at 10, 25 and 50 %, respectively, compared with the control group.

An increase in ear thickness (+1.4%, +1.6% and +13.5%) was noted in animals treated at 10, 25 and 50%, respectively, compared with the control group.

 

Table 7.4.1/1: BrdU index & Stimulation index per group and calculation of EC1.6

Groups

Test item

Brdu-index (Mean*)

Stimulation Index (SI)

Result

1

0 (AOO)

0.455

-

Negative

2

10 %

0.610

1.34

Negative

3

25 %

0.750

1.65

Positive

4

50 %

0.946

2.08

Positive
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test item has to be classified as skin sensitiser category 1B (H317) in accordance with CLP Regulation (EC) No 1272/2008.
Executive summary:

In a local lymph node assay (LLNA:BrdU) performed according to OECD Guideline 442B and in compliance with GLP, groups of CBA/J mice (4 females/dose) were topically applied with 25 µL of the registered substance at concentrations of 10, 25 and 50% to the dorsal surface of both ears for three consecutive days. Vehicle control group of four females received the vehicle (acetone/olive oil (4/1; v/v)). On Day 5, 0.5 mL of BrdU solution (10 mg/mL) was injected by intra-peritoneal route. On Day 6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by measurement of BrdU content in DNA of lymphocyte with ELISA kit and Stimulation Indices (SI) were calculated. Animals were observed for mortality, clinical signs and body weight during the study. The test concentrations for the main study were determined from a preliminary study.

No mortality and no signs of systemic toxicity were noted in the treated and control animals during the test.

Dryness of the skin was noted on Day 6 in all animals treated with the test item at 25 and 50%.

An increase in ear weight (+3%, +27% +47.8%) was noted in animals treated at 10, 25 and 50%, respectively, compared with the control group.

An increase in ear thickness (+1.4%, +1.6% and +13.5%) was noted in animals treated at 10, 25 and 50%, respectively, compared with the control group.

Therefore, the test item has to be considered as not excessively irritant at these concentrations.

Stimulation Index was 1.34, 1.65 and 2.08 for the treated groups at 10, 25 and 50%, respectively. The EC1.6 was estimated to be 22.58% by linear regression.

Based on the EC1.6 of the current positive control, tested with HCA, the positive control is classified as sensitiser, as expected.

Therefore, the test item has to be classified as skin sensitiser category 1B (H317) in accordance with CLP Regulation (EC) No 1272 /2008.

Endpoint:
skin sensitisation: in vivo (LLNA)
Remarks:
In vivo study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
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)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Test Material: Terpenes and terpenoids, turpentine oil, alpha pinene fraction oligomers
Description: extremely viscous fraction oligomers
CAS 70750-57-1
Batch Number: 0910002624
Purity: 100%
Date received: 14 March
Expiration date: Indefintive
Storage: room temperature in the dark
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd. Oxon, UK
- Age at study initiation: 8-12 weeks old
- Weight at study initiation: 15-23 g
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): free access
- Water (e.g. ad libitum): free access
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature : 19-25°C
- Humidity: 30-70%
- Air changes (per hr):
- Photoperiod: 12h dark, 12 h light
Vehicle:
other: butanone
Concentration:
Mice were treated by daily application of 25 μl of the test material at concentration of 25% or 50% w/w in butanone.
No. of animals per dose:
4
Details on study design:
The preliminary test showed no systemic toxicity or excessive local skin irritation at the highest suitable concetration. Daily application of 25µl of the appropriate concentration of the test item was applied to the dorsal surface of each ear for three consecutive days. Five days following the first topical application all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H-methyl thymidine giving a total of 20 µCi to each mouse.
Statistics:
The proliferation response of lymph node was expressed as the number of radioactive disintegrations per minutes per lymph node and as the ratio of 3HTdR incorporation into lymph node cells of the test nodes relative to that recorded for the control nodes. (SI).
Key result
Parameter:
SI
Value:
1.31
Remarks on result:
other: Concentration 5% w/w in butanone
Key result
Parameter:
SI
Value:
1.04
Remarks on result:
other: 10% w/w in butanone
Key result
Parameter:
SI
Value:
2.94
Remarks on result:
other: 25% w/w in butanone
Cellular proliferation data / Observations:
Clinical Observation: Mild (very slight) erythema on the ears was noted in animals treated with the test item at a concetration of 25% w/W in butanone. There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Body weight: One animal, treated with the test ite, at concentration of 10% w/w in butanone, showed a slightly greater than expected bodyweight loss (3g) over the test period. Bodyweight changes of the remaining test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals.
Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item is considered to be a non-sensitiser under the condition of this test.
Executive summary:

A LLNA test was performed to investigate the skin sensitisation potential of Terpenes and terpenoids, turpentine oil, alpha pinene fraction oligomers. The study was performed according to the OECD 429 and under GLP conditions. The test item was applied onto the dorsal surface of the ear of CBA/Ca strain female mice. Following a preliminary screening test in which no systemic toxicity was recorded at concentration of 25% w/w, this concentration was selected as the highest dose investigated. The results were expressed as Simulation Index (SI) and under the condition of the study, the test item was considered to be a non-sensitiser.

Endpoint:
skin sensitisation: in vivo (LLNA)
Remarks:
in vivo study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
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)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test Material: Terpenes and terpenoids, turpentin oil, beta- pinene fraction polymerised
- CAS 70750-52-8
- Batch Number 24 August 2011
- Date received: 14 March 2012
- Expire date: Indefinite
- Storage considtions: room temperature
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd. Oxon, UK
- Age at study initiation: 8-12 weeks old
- Weight at study initiation: 15-23 g
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): free access
- Water (e.g. ad libitum): free access
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature : 19-25°C
- Humidity: 30-70%
- Air changes (per hr):
- Photoperiod: 12h dark, 12 h light
Vehicle:
propylene glycol
Concentration:
25 w/w
No. of animals per dose:
4
Details on study design:
The preliminary test showed no systemic toxicity or excessive local skin irritation at the highest suitable concetration. Daily application of 25µl of the appropriate concentration of the test item was applied to the dorsal surface of each ear for three consecutive days. Five days following the first topical application all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H-methyl thymidine giving a total of 20 µCi to each mouse.
Positive control substance(s):
other: Phenylacetaldehyde (90%)
Key result
Parameter:
SI
Remarks:
Concentration (5 w/w) in propylene glycol
Value:
1.29
Variability:
Not applicable
Remarks on result:
other: Negative
Remarks:
Not sensitiser
Key result
Parameter:
SI
Remarks:
Concentration (10 w/w) in propylene glycol
Value:
1.24
Variability:
Not applicable
Remarks on result:
other: Negative
Remarks:
Not sensitiser
Key result
Parameter:
SI
Remarks:
Concentration (25 w/w) in propylene glycol
Value:
1.23
Variability:
Not applicable
Remarks on result:
other: Negative
Remarks:
Not sensitiser
Cellular proliferation data / Observations:
Clinical Observations and Mortality Data
- There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Bodyweight:
- Boduweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group anumals over the same period.
Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item is considered to be a non-sensitiser under the condition of this test.
Executive summary:

The LLNA test was performed to investigate the skin sensitisation potential of Terpenes and terpenoids, turpentine oil, beta pinene fraction oligomers. The study was performed according the OECD Guideline 429 and under GLP conditions. The test item was applied onto the dorsal surface of the ear of CBA/Ca strain female mice. Following a preliminary screening test in which no systemic toxicity was recorded at concentration of 25% w/w, this concentration was selected as the highest dose investigated. The results were expressed as Simulation Index (SI) and under the condition of the study, the test item was considered to be a non-sensitiser.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16th April-17th May 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
To assess skin sensitisation potential of TM-1301 on albino guinea pig according to OECD 406 guidelines.
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The testing using the guinea pig maximisation test protocol was completed on 17 May 2007. The LLNA 429 was not formally adopted by the OECD until 22 July 2010.
Specific details on test material used for the study:
Test item TM-1301 (considered 100% for the study).
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
Test animals:
Supplied by Centre de Production Animale (F-45160 Olivet).
Acclimatisation period: at least five days.
Age: 5-6 weeks old
Weight: 333g - 451g.

Environmental conditions:
Temperature: 19-24°C.
Relative humidity: 39- 60%.
Diet (ad lib): complete feed made from formulation Extralabo from Pietrement. (frequency not stated, roughly 40g a day).
Water: not stated.
Air changes (per hr) : not stated.
Photoperiod (hrs dark / hrs light) : not stated.
Route:
intradermal and epicutaneous
Vehicle:
other: Preliminary studies: olive oil, liquid paraffin; Main study induction phase: olive oil, physiological saline solution; Main study challenge phase: liquid praffin.
Concentration / amount:
Preliminary studies:
Maximum Non Necrotising Concentration (M. N.N.C) determination:
-Test item injected by intradermal route at the folowing concentrations: 100%, 50%, 25% 12.5%, 6.25% and 2.125% diluted in olive oil.
Pre-Maximum Non-Iritant Concentration (Pre-M.N.I.C) determination:
-Test item applied under an occlusive dressing during 25 hours, at the following concentrations: 100%, 50%, 25% and 12.5% diluted in liquid paraffin.
Maximum Non Irritant Concentration (M.N.I.C.) determination:
-After induction by intradermal injection with olive oil and by epicutaneous application with liquid praffin and a 12-day rest phase, the challenge phase of the test item applied under occlusive dressing for 24hours in a single epicutaneous applications at the following concentrations: 25%, 12.5%, 6.25% and 3.125% diluted in liquid paraffin.
Main study:
1st induction:
-2 intradermal injections of test item diluted at 6.25% in olive oil
-2 intradermal injections of Freund's Complete Adjuvant at 50% diluted in physiological saline solution
-2 intradermal injections of a mixture with equal volumes: Freund's Complete Adjuvant at 50% and test item at 12.5% in olive oil
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
25% and 12.5%
No. of animals per dose:
Negative control: 6 animals.
Treated group: 11.
Positive control substance(s):
yes
Remarks:
The result s of 3 last positive groups (reference substance: alpha-hexylcinnamaldehyde tests 11-13) carried out as method sensibility, were presented in appendix.
Positive control results:
see "any other information on results inc. tables"
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
11
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
11
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
6
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
6
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
12.5%
No. with + reactions:
0
Total no. in group:
11
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
12.5%
No. with + reactions:
0
Total no. in group:
11
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
12.5%
No. with + reactions:
0
Total no. in group:
6
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
12.5%
No. with + reactions:
0
Total no. in group:
6
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
25%
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
Test 13
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
25%
No. with + reactions:
7
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
Test 13

POSITIVE CONTROL RESULTS:

Application date 16/01/2007 (test 13, n=10)

Application date 07/02/2006 (test 12, n=10)

Application date 31/01/2006 (test 11, n=10)

Macroscopic evaluations (reading at 24 and 48 hours) of cutaneous reactions

 Test  Reading Time  Concentration

 Quotations      

  % of Positive Responses >=1

 % of Animals Sensitised
     0  1  2  3    
 Treated group Test 11  24h  50%  3 3  4  0   70%  70%
 Treated group Test 11  48h  50%  3 3  4  0   70%  70%
 Treated group Test 11  25h  25%  5 3  2  0   50%  50%
 Treated group Test 11  48h  25%  7 2  1  0   30%  30%
 Treated group test 12  24h  50%  0 2  5  3   100%  100%
 Treated group test 12  48h  50%  1  4  5  0  90%  90%
 Treated group test 12  24h  25%  1 2  7  0   90%  90%
 Treated group Test 12  48h  25%  1 6  3  0   90% 90% 
 Treated group Test 13  24h  50%  0 1  9  0   100% 100% 
 Treated group Test 13  48h  50%  1  6 3  0   90% 90% 
 Treated group Test 13  24h  25%  0 2  8  0   100% 100% 
 Treated group Test 13  48h 25%   3 7  0  0   70%  70%

In conclusion, in the view of these results, under these experimental conditions, the substance alpha-hexylcinnamaldehyde must be classified as R 43 "may cause sensitisation by skin contact" in accordance with the criteria for classification, labelling and packaging of dangerous substances and preparations of the E.E.C. Directives 67/548, 2001/59 and 99/45. This substance must be characterised by the symbol "Xi" and the warning label "Irritant".

PRELIMINARY STUDIES (Macroscopic evaluations of cutaneous reactions):

MNNC determination:

 Injection  Number of animals  Concentrations               
     100%  50%  25%  12.5%  6.25%  3.125%
 Intradermic injection  C7565  Ne  Ne  Ne  SlNe  0 0 
 Intradermic injection C7566  Ne  Ne  Ne  SlNe  0  0

Ne=necrosis; SlNe=slight necrosis

No necrosis has been observed, since the concentreation 6.25% and below, the first induction was carried out by intradermal injection was carried at the same concentration.

Maximal Non Necrotising Concentration (MMNC)=6.25%

Pre-MNIC determination:

 Application  Numbe of animals        Concentrations  
     100% 50%  25%  12.5% 
 Topical application under occlusive dressing  C7565  2(E)  0  0 0 
 Toppical application undr occlusive dressing  C7566  2(E)  2(E)  0  0

E=erythema

24 hours after the removal of the occlusive dressing, it was recorded a well defined erythem on the treated area at 100% in the two animals and well defined erythema on the treated area at 50% in one animal. In view of these results, the concentration selected was 100% for the 2nd induction of the main study and the MNIC determination began at this concentration of 25%

MNIC determination:

 Application  Number of animal           Concentrations
     25% 12.5%  6.25%  3.25% 
 Toppical application under occlusive dressing  C7569  0 0  0  0 
 Toppical application under occlusive dressing  C7570  0 0 0  0 
 Toppical application under occlusive dressing  C7571  0 0  0  0 

24 hours after removal of the occlusive dressing, no cutaneous reaction was recorded whatever the tested concentration. In view of this result, the concentrations selected 25% (MNIC) and 12.5% (1/2 MNIC) for the challenge phase.

MAIN STUDY:

Induction phase:

The induction phase was performed by intradermal injection at D0 with the test item at 6.25% in olive oil and epicutaneous application at D7 with the test item at 100% during 48h.

Challenge phase:

The test item has been used at 25% (MNIC) and 12.5% (1/2 MNIC) in liquid praffin.

Overall results (Erythema and Oedema):

Macroscopic evalution (reading at 24 and 48 hours) of cutaneous reactions

 Groups  Reading time  Concentrations           Quotations  % of Positive Responses >=1  % of Animals Sensitised
       0 1 2 3 or >   
 Negative control group  24h  25%  6  0 0  0   0% 0% 
   48h  25%  6 0  0  0   0%  0%
   24h  12.5%  6 0  0  0   0%  0%
   48h  12.5%  6 0  0  0   0%  0%
 Treated Group  24h  25%  11 0  0  0  0%  0% 
   48h  25% 11  0 0  0  0%  0% 
   24h  12.5%  11 0  0  0  0%  0% 
   48h  12.5%  11 0  0  0  0%  0% 

Grading scales:

Erythema: 0=no visible modification, 1=slight or patches of erythema, 2=moderate confluent erythema, 3=internal erythema and swelling.

Oedema: 0=no visible modification, 1=slight oedema, 2=moderate oedema, 3=important oedema.

Individual results:

Macroscopic evaluation (reading at 24 and 48 hours) of cutaneous reaction

 Number of animals  M.N.I.C. (25%)           1/2 M.N.I.C. (12.5%)         
   24h      48h   24h 48h    
 Negative Control group  Er Oe   Er  Oe  Er  Oe  Er  Oe  
 C7575 F  0 0  0  0  0  0  0  0   
 C7576 F  0 0  0  0  0  0  0  0   
 C7577 F  0 0  0  0  0  0  0  0   
 C7578 F  0 0  0  0  0  0  0  0   
 C7579 F  0 0  0  0  0  0  0  0   
 C7580 F  0 0  0 0  0  0  0  0   
 Treated Group  Er  Oe  Er  Oe  Er  Oe  Er  Oe  
 C7581 F  0 0  0  0  0  0  0  0   
 C7582 F  0 0 0  0  0  0  0  0   
 C7583 F  0 0  0  0  0  0  0  0   
 C7584 F  0 0  0  0  0  0  0  0   
 C7585 F  0 0  0  0  0  0  0  0   
 C7586 F  0 0  0  0  0  0  0  0   
 C7587 F  0 0  0  0  0  0  0  0   
 C7588 F  0 0  0  0  0  0  0  0   
 C7589 F  0 0  0  0  0  0  0  0   
 C7590 F  0 0  0  0  0  0  0  0   
 C7591 F  0 0  0  0  0  0  0  0   
 C7582  0 0  0  0  0  0  0  0   

Er=erythema, Oe=oedema, F=female.

No macroscopic cutaneous reactions were recorded in negative control and treated groups in either concentrations.

Weight evolution:

Not any abnormality was recorded in body weight gain of negative control animals and treated animals.

Interpretation of results:
GHS criteria not met
Conclusions:
The study was performed according to the Magnusson and Kligman method and in accordance with O.E.C.D. Guideline N° 406 of July 17th, 1992, and the test method B.6 of the 96/54 E.E.C. Directive and therefore considered to be of the highest quality (reliability Klimisch 1). In view of the study results, under these experimental conditions, the test item TM-1301 must not be classified as, in accordance with the criteria for classification, packaging and labelling of dangerous substances and preparations of the E.E.C. Directives 67/548, 2001/59 and 99/45. No symbol and risk phrase are required.
Executive summary:

After indcution (intradermal injection at 6.25% and epicutaneous application at 100%) of 11 guinea pigs (female) in treated group with the test item TM-1301 and an 11-day rest period, the challenge phase, under an occlusive dressing for 24 hours, consisted to a single topical application of the test item diluted at 25% and at 12.5% in liquid paraffin, according to the experimental protocol established from the O.E.C.D. guideline no. 406 dated July 17th 1992, and the methods B.6 of the E.C no. 96/54 dated July 30th 1996. No macroscopic cutaneous reactions attributable to allergy was recorded during the examination following the removal of occlusive dressing (challenge phase) from the animals in the treated group with the test item. No cutaneous intolerence was recorded from the animals from the negative control group. In conclusion, in view of these results, the test item TM-1301 must not be classified, in accordance to classification, packaging and labelling of dangerous substances and preparations of the E.C. Directives 67/548. 2001/59 and 99/45. No symbol or risk phrase are required.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In conclusion, alpha-pinene and beta-pinene oligomers are considered not to have skin sensitisation potential and subsequently does not require classification for skin sensitisation potential accordingly to EC No. 1272/2008. 

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Alpha-pinene and beta-pinene oligomers are considered not to have skin sensitisation potential and subsequently does not require classification for skin sensitisation potential accordingly to EC No. 1272/2008.