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Diss Factsheets

Administrative data

Description of key information

In vivo skin sensitisation. Weight of evidence. Read-across approach: Popliteal lymph node assay (PLNA). Based on the read-across approach from the analogue alpha terpineol, laevo alpha terpineol can be considered as non sensitiser.

In vivo skin sensitisation. Weight of evidence. Read-across approach: Modified FCA-method (Freund’s complete adjuvant) similar to OECD 406 Guideline. Based on the read-across approach from the analogue alpha terpineol, laevo alpha terpineol can be considered as non sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: Rat popliteal lymph node assay (PLNA). Although the predictive value of PLNA was initially focused on auto-immune-like reactions, more recently it has been regarded as a screening test useful for detecting a broader class of sensitizing or immuno-stimulating chemicals (Pieters and Albers, 1999; Ravel and Descotes, 2005). In this study, the rat PLNA was used to evaluate the immuno-sensitizing potential of 10 monoterpenes found in the essential oils of a variety of aromatic, edible and medicinal plants.

GLP compliance:
not specified
Type of study:
other: Popliteal lymph node assay (PLNA)
Justification for non-LLNA method:
The PLNA seems to be a reliable test for screening chemicals causing sensitization via routes of exposure other than the skin (Goebel et al., 1996; Tuschl et al., 2002).
Species:
other: rat
Strain:
other: Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Oswaldo Cruz Foundation (FIOCRUZ) breeding stock
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7–8 week-old
- Weight at study initiation: 145 ± 23 g
- Housing: All rats were individually housed in standard plastic cages with stainless steel cover lids and wood shavings as bedding.
- Diet (e.g. ad libitum): ad libitum (Nuvitall, Nuvilab, Curitiba, PR, Brazil)
- Water (e.g. ad libitum): ad libitum (tap water)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1ºC,
- Humidity (%): 70%
- Photoperiod (hrs dark / hrs light): 12-h dark/light cycle
Route:
other: subcutaneous
Vehicle:
DMSO
Concentration / amount:
5 mg/paw of test substance (50 µL of test substance in vehicle)
Day(s)/duration:
7 days
Adequacy of induction:
other: It is of note that, as a rule, 5 mg/paw has been the highest dose of a test compound assayed in the rat PLNA (Vial et al., 1997; Descotes et al., 1997).
No. of animals per dose:
10
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: one per animal
- Exposure period: Seven days after footpad injection, rats were killed by CO2 inhalation
- Test groups: one treated group (10 animals)
- Control group: barbital (negative control), saline (vehicle control for negative) and DMSO (vehicle control for treated group)
- Site: right footpad (treated group). The contralateral (left) hind footpad was used as the control and thus it was injected with 50 µL of vehicle alone. When the vehicles were tested, the right hind footpad was injected with 50 µL of DMSO or saline while the left hind footpad remained untreated.

OTHER:
Seven days after footpad injection, rats were killed by CO2 inhalation. The popliteal lymph nodes (PLN) were carefully removed, placed in phosphate buffer saline (PBS), freed from adherent fatty tissue and weighed. PLNs were then transferred to a glass tube where cells were extracted by agitation and gentle grinding using a micropestle and suspended in a known volume of PBS on ice. PLN cell number (cellularity) was counted using a Neubauer chamber. Results were expressed as weight
(WI) and cellularity (CI) indices. WI and CI were calculated by dividing the value (weight or cellularity) obtained for the treated (right) side PLN by that obtained for the control (left) side PLN.

Positive control substance(s):
yes
Remarks:
(chlorpromazine)
Positive control results:
see table on "any other information on results incl. tables"
Key result
Reading:
1st reading
Group:
test chemical
Dose level:
5 mg
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Group:
negative control
Dose level:
5 mg
No. with + reactions:
0
Total no. in group:
8
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Group:
positive control
Dose level:
5 mg
Remarks on result:
positive indication of skin sensitisation

Table 1: Primary PLNA responses

 Criteria

Negative controls

Vehicle

Chlorpromazine

 

 Terpineol 

 Barbital 

DMSO

 Saline

0.5 mg/paw

2.5 mg/paw

5.0 mg/paw

 N 

 10 

 8 

 47 

 50 

 4 

 6 

 11 

 WI 

 1.32 ± 0.71 

 1.06 ± 0.36 

 1.48 ± 0.65 

 1.12 ± 0.90 

 1.30 ± 0.35 

 2.06 ± 0.81* 

 3.22 ± 1.13* 

 CI 

 2.00 ± 3.32 

 1.34 ± 0.92 

 2.95 ± 3.68 

 2.04 ± 2.03 

 1.26 ± 0.81 

 8.49 ± 8.91* 

 8.28 ± 8.19* 

 IPR, no. (%) 

 2 (20) 

 0 (0) 

 5 (10.6) 

 3 (6) 

 0.0 

 50.0 

 63.6* 

* indicates that the value differs from that of the lowest dose group (0.5 mg/paw)

- Injected doses were 5 mg/paw (terpineol and barbital) or 50 µL /paw (vehicles).

- Values are means ± SD;

- Weight (WI) and Cellularity (CI) indices: values for the draining popliteal lymph node of the treated (right paw) side divided by that of the control (left paw) side.

- IPR, number (%) of rats within the group with WI ≥ 2 and CI ≥ 5.

- Terpineol was classified as negative because group mean values for WI and CI were lower than 2 and 5, respectively.

Interpretation of results:
other: No category (CLP Regulation EC no. 1272/2008)
Conclusions:
Under these test conditions, alpha-terpineol was not considered as a sensitiser.
Executive summary:

In a popliteal lymph node assay (PLNA), a group of 10 female Wistar rats were injected subcutaneously with alpha terpineol at 5 mg/paw into the right hind footpad while the contralateral footpad was injected with the vehicle (DMSO) alone. Chlorpromazine (CPZ) and barbital were used as positive and negative controls, respectively. Weight (WI) and cellularity (CI) indices for draining PLNs were determined 7 days after treatment. Weight (WI) and cellularity (CI) indices for alpha terpineol was determined to be 1.32 and 2.00, respectively. Alpha terpineol was classified as negative because group mean values for WI and CI were lower than 2 and 5, respectively. Under these test conditions, alpha terpineol was not considered as a sensitiser.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
modified FCA-method (Freund’s complete adjuvant) (Hausen and Angel, 1992)
Deviations:
yes
Remarks:
(no details of housing and evironmental conditions of the animals)
GLP compliance:
not specified
Type of study:
Freund's complete adjuvant test
Justification for non-LLNA method:
Test was performed before OECD LLNA guideline approval and based on reliable method according to OECD 406 guideline.
Species:
guinea pig
Strain:
other: Pirbright-white stem
Sex:
female
Route:
epicutaneous, open
Vehicle:
physiological saline
Concentration / amount:
30 mg of oxidized tea tree oil (TTO) dissolved in 4 mL of FCA and emulsified with 4 mL of physiological saline.
Day(s)/duration:
11 days
Adequacy of induction:
not specified
No.:
#1
Route:
epicutaneous, open
Vehicle:
other: acetone
Concentration / amount:
0.05 mL of 10% alpha terpineol-solution in acetone
Adequacy of challenge:
not specified
No. of animals per dose:
10
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1
- Exposure period: 11 days
- Test groups: one treated group (10 animals)
- Concentrations: 30 mg of oxidized tea tree oil (TTO) dissolved in 4 mL of FCA and emulsified with 4 mL of physiological saline.

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Test groups: one treated group (10 animals)
- Site: clipped and shaved right flank of the animals.
- Concentrations: 10% alpha terpineol-solution in acetone
- Evaluation (hr after challenge): 24, 48 and 72 hours (according to the rules of the International Contact Dermatitis Research Group (ICDRG)).


Positive control substance(s):
no
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0.05 mL of 10% alpha terpineol-solution in acetone
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0.05 mL of 10% alpha terpineol-solution in acetone
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Group:
negative control
Remarks on result:
other: no data
Key result
Reading:
1st reading
Group:
positive control
Remarks on result:
other: No data

Table 1: Results of the Sensitization and Challenging Experiments

Nº of guinea pigs

Sensitized with

Challenged with

Concentration

Mean response

24 h

48 h

10

TTO, oxidized

Alpha terpineol

10%

0

0
Interpretation of results:
other: No category (CLP Regulation EC no. 1272/2008)
Conclusions:
Under these test conditions, alpha-terpineol was found to be a non skin sensitiser.

Executive summary:

Experimental sensitization was performed in albino guinea pigs by a modified FCA-method (Freund’s complete adjuvant). For induction, 30 mg of oxidized tea tree oil (TTO) dissolved in 4 mL of FCA and emulsified with 4 mL of physiological saline were used on 10 female guinea pigs. Eleven days after induction, open epicutaneous challenge was performed by application of 0.05 mL of 10% alpha terpineol-solution in acetone on the clipped and shaved right flank of the animals. The reactions were read at 24 hours, 48 hours, and 72 hours. As the maximum reaction was observed in most of the animals already in the 48 hour reading, the 72 hour readings were not noted. The mean responses obtained after 24 h and 48 h were 0 and thus it is concluded that alpha terpineol is not a skin sensitizer.

In a popliteal lymph node assay (PLNA), a group of 10 female Wistar rats were injected subcutaneously with alpha terpineol at 5 mg/paw into the right hind footpad while the contralateral footpad was injected with the vehicle (DMSO) alone. Chlorpromazine (CPZ) and barbital were used as positive and negative controls, respectively. Weight (WI) and cellularity (CI) indices for draining PLNs were determined 7 days after treatment. Weight (WI) and cellularity (CI) indices for alpha terpineol was determined to be 1.32 and 2.00, respectively. Alpha terpineol was classified as negative because group mean values for WI and CI were lower than 2 and 5, respectively. Under these test conditions, alpha terpineol was not considered as a sensitiser.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with column 2 of REACH Annex VII, the study does not need to be conducted since an in vivo study on sensitisation is available.
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance alpha terpineol which shares the same functional groups with the substance L-alpha terpineol also has comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Key result
Reading:
1st reading
Group:
test chemical
Dose level:
5 mg
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Remarks:
(read-across from an analogue for which no indication of skin sensitisation was obtained with 5 mg tested)
Key result
Reading:
1st reading
Group:
negative control
Dose level:
5 mg
No. with + reactions:
0
Total no. in group:
8
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Group:
positive control
Dose level:
5 mg
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
other: No category (CLP Regulation EC no. 1272/2008)
Conclusions:
Based on the read-across approach from the analogue alpha terpineol, laevo alpha terpineol can be considered as non sensitiser.
Executive summary:

In a popliteal lymph node assay (PLNA), a group of 10 female Wistar rats were injected subcutaneously with alpha terpineol at 5 mg/paw into the right hind footpad while the contralateral footpad was injected with the vehicle (DMSO) alone. Chlorpromazine (CPZ) and barbital were used as positive and negative controls, respectively. Weight (WI) and cellularity (CI) indices for draining PLNs were determined 7 days after treatment. Weight (WI) and cellularity (CI) indices for alpha terpineol was determined to be 1.32 and 2.00, respectively. Alpha terpineol was classified as negative because group mean values for WI and CI were lower than 2 and 5, respectively. Under these test conditions, alpha terpineol was not considered as a sensitiser. Based on these results, the read-across approach was applied and laevo alpha terpineol can be considered as non sensitizer.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance alpha terpineol which shares the same functional groups with the substance L-alpha terpineol also has comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0.05 mL of 10% alpha terpineol-solution in acetone
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Remarks:
(read-across from an analogue for which no indication of skin sensitisation was obtained when tested with 0.05 mL of 10% test item-solution in acetone)
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0.05 mL of 10% alpha terpineol-solution in acetone
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Remarks:
(read-across from an analogue for which no indication of skin sensitisation was obtained when tested with 0.05 mL of 10% test item-solution in acetone)
Key result
Reading:
1st reading
Group:
negative control
Remarks on result:
other: no data
Key result
Reading:
1st reading
Group:
positive control
Remarks on result:
other: No data
Interpretation of results:
other: No category (CLP Regulation EC no. 1272/2008)
Conclusions:
Based on the read-across approach from the analogue alpha terpineol, laevo alpha terpineol can be considered as non skin sensitiser.

Executive summary:

Experimental sensitization was performed with alpha terpineol in albino guinea pigs by a modified FCA-method (Freund’s complete adjuvant). For induction, 30 mg of oxidized tea tree oil (TTO) dissolved in 4 mL of FCA and emulsified with 4 mL of physiological saline were used on 10 female guinea pigs. Eleven days after induction, open epicutaneous challenge was performed by application of 0.05 mL of 10% alpha terpineol-solution in acetone on the clipped and shaved right flank of the animals. The reactions were read at 24 hours, 48 hours, and 72 hours. As the maximum reaction was observed in most of the animals already in the 48 hour reading, the 72 hour readings were not noted. The mean responses obtained after 24 h and 48 h were 0 and thus it is concluded that alpha terpineol is not a skin sensitizer. Based on these results, the read-across approach was applied and laevo alpha terpineol can be considered as non sensitizer.

In a popliteal lymph node assay (PLNA), a group of 10 female Wistar rats were injected subcutaneously with alpha terpineol at 5 mg/paw into the right hind footpad while the contralateral footpad was injected with the vehicle (DMSO) alone. Chlorpromazine (CPZ) and barbital were used as positive and negative controls, respectively. Weight (WI) and cellularity (CI) indices for draining PLNs were determined 7 days after treatment. Weight (WI) and cellularity (CI) indices for alpha terpineol was determined to be 1.32 and 2.00, respectively. Alpha terpineol was classified as negative because group mean values for WI and CI were lower than 2 and 5, respectively. Under these test conditions, alpha terpineol was not considered as a sensitiser.

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

In vivo skin sensitisation. Weight of evidence. Read-across approach: In a popliteal lymph node assay (PLNA), a group of 10 female Wistar rats were injected subcutaneously with alpha terpineol at 5 mg/paw into the right hind footpad while the contralateral footpad was injected with the vehicle (DMSO) alone. Chlorpromazine (CPZ) and barbital were used as positive and negative controls, respectively. Weight (WI) and cellularity (CI) indices for draining PLNs were determined 7 days after treatment. Weight (WI) and cellularity (CI) indices for alpha terpineol was determined to be 1.32 and 2.00, respectively. Alpha terpineol was classified as negative because group mean values for WI and CI were lower than 2 and 5, respectively. Under these test conditions, alpha terpineol was not considered as a sensitiser. Based on these results, the read-across approach was applied and laevo alpha terpineol can be considered as non sensitizer.

In vivo skin sensitisation. Weight of evidence. Read-across approach: Experimental sensitization was performed with alpha terpineol in albino guinea pigs by a modified FCA-method (Freund’s complete adjuvant). For induction, 30 mg of oxidized tea tree oil (TTO) dissolved in 4 mL of FCA and emulsified with 4 mL of physiological saline were used on 10 female guinea pigs. Eleven days after induction, open epicutaneous challenge was performed by application of 0.05 mL of 10% alpha terpineol-solution in acetone on the clipped and shaved right flank of the animals. The reactions were read at 24 hours, 48 hours, and 72 hours. As the maximum reaction was observed in most of the animals already in the 48 hour reading, the 72 hour readings were not noted. The mean responses obtained after 24 h and 48 h were 0 and thus it is concluded that alpha terpineol is not a skin sensitizer. Based on these results, the read-across approach was applied and laevo alpha terpineol can be considered as non sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, the substance does not need to be classified for skin sensitization according to CLP Regulation no. 1272/2008.