Essential oil of Mentha Spicata L., Erospicata (Labiatae) obtained from aerial parts by steam distillation

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 - 26 Oct 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Version / remarks:

adopted 22 July 2010

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Test material information

Constituent 1

Reference substance name:

Essential oil of Mentha Spicata L., Erospicata (Labiatae) obtained from aerial parts by steam distillation

EC Number:

811-616-1

Cas Number:

1563063-07-9

Molecular formula:

not applicable for UVCB substances

IUPAC Name:

Essential oil of Mentha Spicata L., Erospicata (Labiatae) obtained from aerial parts by steam distillation

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/N

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: 9 weeks
- Weight at study initiation: 17.6 - 21.9 g (range-finding study), 17.3 - 22.9 g (main study part 1), 17.9 - 20.6 g (main study part 2)
- Housing: 2 - 3 animals per cage in polysulfone cages (200W x 320D x 140H mm)
- Diet: Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C (Envigo RMS. Ltd., USA), ad libitum
- Water: public tap water filtered and irradiated by ultraviolet light, ad libitum
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 - 23.0
- Humidity (%): 45.8 - 58.5
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Range-finding study:
5, 10, 25, 50% (w/v) and 100%

Main study:
1, 10% (w/v) and 100% (Part 1)
25 and 50% (Part 2)

No. of animals per dose:

2 (range-finding study), 5 (main study)

Details on study design:

RANGE-FINDING STUDY:
- Compound solubility: The test substance was dissolved in AOO in a preliminary solubility test. Therefore, AOO was utilized as vehicle for this study.
Dose selection was based on the consecutive doses and dose levels are selected from a series of appropriate concentrations such as 5, 10, 25, 50 and 100%.The preliminary screening test was performed under identical conditions as the main study, except determination of stimulation indices.
- Irritation: Excessive local irritation is indicated by an erythema score ≥ 3 on any day of measurement.
- Ear thickness: Ear thickness was ≥ 25% on any day of measurement.
- Systemic toxicity: The following clinical observations indicate systemic toxicity when used as part of an integrated assessment and therefore may indicate the maximum dose level to use in the main study: changes in nervous system, changes in behavior, changes in respiratory patterns, and changes in food and water consumption.
Based on the results of the dose range finding study the highest dose for the main study was selected as 100%. Two additional low dose levels (10 and 1%) were included after discussion with the sponsor/ study monitor (Part 1). Two additional mid dose levels (50 and 25%) were included after discussion with the sponsor/ study monitor (Part 2). In addition, the positive and negative control groups were included in the main study.

MAIN STUDY
- Name of test method: 5-bromo-2-deoxyuridine (BrdU) incorporation determined by ELISA
- Criteria used to consider a positive response: A stimulation index (SI) was calculated for each group using the BrdU labelling index of each test group divided by the BrdU labelling index of the vehicle control group. A test substance is considered positive if the SI value is ≥ 1.6.

TREATMENT PREPARATION AND ADMINISTRATION: The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance on Day 1. The application was repeated on Day 2 and 3. Two days after the third application on Day 5, an intraperitoneal injection of 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) was made. Approximately 24 h later, following injection of BrdU, the mice were sacrificed and draining auricular lymph nodes were excised and pooled for each individual animal. A single cell suspension was prepared by separation through a nylon mesh. In each case, the target volume of the cell suspension was adjusted to the determined optimized volume. The optimized volume was based on the mean absorbance within 0.1 - 0.2 in the vehicle control group. BrdU was measured by ELISA using a commercial kit. Briefly, 100 µL of the cell suspension was added to the wells of a microplate in triplicate. After fixation and denaturation of the cell suspension, anti-BrdU antibody was added to each well. Subsequently, anti-BrdU antibody was removed by washing and the substrate solution was added. Absorbance was measured at 370 nm with a reference wavelength of 492 nm.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Statistical analysis was conducted using a statistical program (version 9.3, SAS Institute Inc., U.S.A.) for the data including body weight, erythema score, ear thickness, ear weight and stimulation index. Bartlett’s test was employed on homogeneity of variance (significance level: 0.05) for body weights, ear thickness, ear weight and stimulation index data. One-way analysis of variance (ANOVA) was employed on homogeneous data. Dunnett’s t-test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group and each of the test substance groups or positive substance group. Since homoscedasticity was rejected, Kruskal-Wallis test was employed on heterogeneous data and Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group and each of the test substance groups or positive substance group. Kruskal-Wallis test for the erythema score was employed on heterogeneous data, and Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group and each of the test substance groups or positive substance group.

Results and discussion

Positive control results:

Body weight: Mean body weights for Day 1 and Day 6 were 20.0 and 20.3 g, respectively. There were no significant differences when compared to the negative control group.
Ear thickness: Mean ear thickness was 0.20 and 0.23 mm on Day 1 and Day 6, respectively.
Ear weight: Mean ear punch weight was 15.1 mg. There were significant increases when compared to the negative control group (p<0.05).
Erythrema score: Mean erythema score was 0–2.
Stimulation index: SI value calculated for the positive control was 3.73. There was a significant increase when compared to the negative control group (p<0.01).

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.03

Test group / Remarks:

1% test substance

Results 2

Key result

Parameter:

SI

Value:

1.01

Test group / Remarks:

10% test substance

Results 3

Key result

Parameter:

SI

Value:

2.17

Test group / Remarks:

25% test substance

Results 4

Key result

Parameter:

SI

Value:

3.06

Test group / Remarks:

50% test substance

Results 5

Key result

Parameter:

SI

Value:

3.09

Test group / Remarks:

100% test substance

Results 6

Key result

Parameter:

other: EC1.6

Value:

16.7

Cellular proliferation data / Observations:

IRRITATION, EAR THICKNESS and EAR WEIGHTS:
Erythema score:
Part 1: In the negative control group, the mean erythema score was 0 and 0 on Day 1 and Day 6 after dosing, respectively.
In the test substance groups at 1, 10 and 100%, the mean erythema scores were 0–0, 0–0 and 0–2, respectively.
However, since the erythema score was below 3, the value is not considered to be excessive for local irritation.
Part 2: In the negative control group, the mean erythema score was 0 and 0 on Day 1 and Day 6 after dosing, respectively.
In the test substance groups at 25 and 50%, the mean erythema scores were 0–0 and 0–0, respectively. There were no significant differences when compared to the negative control group.

Ear thickness:
Part 1: In the negative control group, the mean ear thickness was 0.18 and 0.19 mm on Day 1 and Day 6 after dosing, respectively.
In the test substance groups at 1, 10 and 100%, the mean ear thickness values (Day 1 and Day 6) were 0.19 and 0.19, 0.19 and 0.20 as well as 0.19 and 0.21 mm, respectively. There were significant increases when compared to the negative control group (p<0.05: Day 1 (100%), p<0.01: Day 3 (1, 10 and 100%), Day 6 (10 and 100%)). However, since the increase in ear thickness was below 25%, the value is not considered to be excessive for local irritation.
Part 2: In the negative control group, the mean ear thickness was 0.19 and 0.19 mm on Day 1 and Day 6 after dosing, respectively.
In the test substance groups at 25 and 50%, the mean ear thickness values (Day 1 and Day 6) were 0.19 and 0.20 as well as 0.19 and 0.20 mm, respectively. There were significant increases when compared to the negative control group (p<0.05: Day 6 (25%), p<0.01: Day 6 (50%)). However, since the increase in ear thickness was below 25%, the value is not considered to be excessive for local irritation.

Ear weights:
Part 1: In the negative control group, the mean ear punch weight was 12.5 mg.
In the test substance groups at 1, 10 and 100%, the mean ear punch weights were 12.6, 12.8 and 12.7 mg, respectively. There were no significant differences when compared to the negative control group.
Part 2: In the negative control group, the mean ear punch weight was 13.3 mg.
In the test substance groups at 25 and 50%, the mean ear punch weights were 13.8 and 14.2 mg, respectively. There were no significant differences when compared to the negative control group.


EC1.6 CALCULATION: EC1.6 =2^{log2(c) + (3-d)/(b-d) x [log2(a)-log2(c)]}
a = The dose concentration with higher SI; b = The higher SI value, c = The dose concentration with lower SI; d = the lower SI value
EC1.6 = 16.7%


CLINICAL OBSERVATIONS:
Part 1 and 2: There were no abnormal clinical signs or deaths in any dosing group during the observation period.

BODY WEIGHTS:
Part 1: In the negative control group, the mean body weights were 19.7 and 19.5 g on Day 1 and Day 6 after dosing, respectively.
In the test substance groups at 1, 10 and 100%, the mean body weights (Day 1 and Day 6) were 20.0 and 19.8, 19.4 and 20.3 as well as 20.1 and 19.7 g, respectively. There were no significant differences when compared to the negative control group.
Part 2: In the negative control group, the mean body weights were 19.4 and 19.3 g on Day 1 and Day 6 after dosing, respectively.
In the test substance groups at 25 and 50%, the mean body weights (Day 1 and Day 6) were 18.7 and 18.1 as well as 18.6 and 18.2 g, respectively. There were no significant differences when compared to the negative control group.

Applicant's summary and conclusion

Interpretation of results:

other: Skin Sens Cat 1B according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the local lymph node assay, the test substance revealed a SI ≥ 1.6 at concentrations of 25, 50 and 100%. The calculated EC1.6 value was 16.7%. Therefore, the test substance is considered as weak sensitiser.