p-menth-1-en-8-ol

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

Assessment of sensitization potential of monoterpenes using the rat popliteal lymph node assay

Author:

Friedrich K

Year:

2007

Bibliographic source:

Food and Chemical Toxicology 45 (2007) 1516–1522

Materials and methods

Test guideline

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).

Test material

Test material information

Constituent 1

Reference substance name:

p-menth-1-en-8-ol

EC Number:

202-680-6

EC Name:

p-menth-1-en-8-ol

Cas Number:

98-55-5

Molecular formula:

C10H18O

IUPAC Name:

2-(4-methylcyclohex-3-en-1-yl)propan-2-ol

In vivo test system

Test animals

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

Study design: in vivo (non-LLNA)

Induction

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)

Results and discussion

Positive control results:

see table on "any other information on results incl. tables"

In vivo (non-LLNA)

Results

Results 1

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

Results 2

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

Results 3

Key result

Reading:

1st reading

Group:

positive control

Dose level:

5 mg

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.