Ocimum basilicum, ext.

Endpoint summary

Description of key information

Skin sensitisation (OECD TG 429): Sensitising (EC3 of <2.5%)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

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)

Version / remarks:

The assays were conducted according to the method of Kimber et al. (1992, 1994) as formalized in OECD Guideline 429 (OECD, 2002)

Deviations:

no

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Interfauna UK, Shaw's Farm, Blackthorne, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 17–21 g
- Housing: 4 animals per cage
- Diet (e.g. ad libitum): ad libitum (Porton Combined Diet, pelleted diet; Special Diets Services Ltd., Witham, United Kingdom)
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19–25
- Humidity (%): 30–70
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

other: EtOH:DEP 1:3

Concentration:

2.5%, 5%, 10%, 25% or 50% w/v

No. of animals per dose:

4

Details on study design:

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: For each concentration of test material, a stimulation index (SI) relative to the concurrent vehicle treated control was calculated. The SI value for each test material was calculated by dividing the mean dpm at a given dose level by the mean dpm of the vehicle control group. A material was considered a sensitizer if at least one concentration of the test material was observed to have an SI value of 3 or more.

ADMINISTRATION
- Test item: 25 µL of the test substance or vehicle control was applied to the back of the ear in 4 female mice per dose group. Dosing occurred daily for three consecutive days.
- 3H-Methyl Thymidine: on day 6 after the first application, the animals were injected intravenously (tail vein) with 250 µL phosphate buffered saline (PBS) containing 20 µCi of [3H] methyl thymidine.

TERMINATION
Five hours later, the mice were euthanized and the draining auricular lymph nodes were excised and pooled for each experimental group. Suspensions of the lymph node cells were prepared (200-mesh stainless steel gauze). The cell suspensions were washed thrice in PBS and precipitated overnight at 4 °C with 5% w/v trichloroacetic acid (TCA). The samples were pelleted by centrifugation, and resuspended in 1 ml of TCA. The incorporation of 3HTdR was measured by by scintillation counting and expressed as disintegrations per minute (dpm) per lymph node for each experimental group.

Positive control substance(s):

not specified

Statistics:

The EC3 value was taken as a measure of relative sensitization potential for each material. Using two data points on the dose response curve, one immediately above and one below the SI value of three, the EC3 value was calculated utilizing the following equation presented by Basketter et al. (1999): EC3 = c+[(3-d)/(b-a)*(a-c) where the data points lying directly above and below the SI value of 3 on the dose–response curve have the coordinates (a,b) and (c,d), respectively.

Positive control results:

Not specified

Key result

Parameter:

EC3

Remarks:

%

Value:

< 2.5

Variability:

not specified

Test group / Remarks:

2.5% test substance

Parameter:

SI

Value:

3

Variability:

not specified

Test group / Remarks:

2.5% test substance

Parameter:

SI

Value:

3

Variability:

not specified

Test group / Remarks:

5% test substance

Parameter:

SI

Value:

8

Variability:

not specified

Test group / Remarks:

10% test substance

Parameter:

SI

Value:

17.6

Variability:

not specified

Test group / Remarks:

25% test substance

Parameter:

SI

Value:

25.2

Variability:

not specified

Test group / Remarks:

50% test substance

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Mean DPM at 0 (vehicle), 2.5, 5, 10, 25, and 50% were 334, 1011, 1013, 2689, 5896 and 8434 respectively.

DETAILS ON STIMULATION INDEX CALCULATION
The SI value for each test material was calculated by dividing the mean dpm at a given dose level by the mean dpm of the vehicle control group.

EC3 CALCULATION
The EC3 value, or estimated concentration of test material required to elicit an SI of 3 or more, was derived from the dose–response data by linear interpolation.

Interpretation of results:

other: Skin sensitiser

Remarks:

in accordance with EU CLP (EC 1272/2008 and its updates)

Conclusions:

Under the conditions of this study, the test item was considered to be a sensitiser based on a derived EC3 value of <2.5%.

Executive summary:

The skin sensitisation potential of the test substance has been tested according to the OECD TG 429 (Local Lymph Node Assay) guideline. An amount of 25 µL of the test substance (at 2.5, 5, 10, 25 and 50%) or vehicle control was applied to the back of the ear in 4 female mice per dose group. Dosing occurred daily for three consecutive days. On day 6 after the first application, the animals were injected intravenously (tail vein) with 250 µL phosphate buffered saline (PBS) containing 20 µCi of [3H] methyl thymidine. Five hours later, the mice were euthanized and the draining auricular lymph nodes were excised and pooled for each experimental group. Suspensions of the lymph node cells were prepared (200-mesh stainless steel gauze). The cell suspensions were washed three times in PBS and precipitated overnight at 4 °C with 5% w/v trichloroacetic acid (TCA). The samples were pelleted by centrifugation, and resuspended in 1 ml of TCA.  The incorporation of 3HTdR was measured by scintillation counting and expressed as disintegrations per minute (dpm) per lymph node for each experimental group. Stimulation Indices of 3, 3, 8, 17.6 and 25.2 were observed at concentrations of 2.5, 5, 10, 25 and 50% respectively. The EC3 was calculated to be <2.5% and no NOEC could be derived. Based on these results the substance is considered to be a skin sensitiser.

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin sensitisation study (LLNA)

The skin sensitisation potential of the test substance has been tested according to the OECD TG 429 (Local Lymph Node Assay) guideline. An amount of 25 µL of the test substance (at 2.5, 5, 10, 25 and 50%) or vehicle control was applied to the back of the ear in 4 female mice per dose group. Dosing occurred daily for three consecutive days. On day 6 after the first application, the animals were injected intravenously (tail vein) with 250 µL phosphate buffered saline (PBS) containing 20 µCi of [3H] methyl thymidine. Five hours later, the mice were euthanized and the draining auricular lymph nodes were excised and pooled for each experimental group. Suspensions of the lymph node cells were prepared (200-mesh stainless steel gauze). The cell suspensions were washed three times in PBS and precipitated overnight at 4 °C with 5% w/v trichloroacetic acid (TCA). The samples were pelleted by centrifugation, and resuspended in 1 ml of TCA.  The incorporation of 3HTdR was measured by scintillation counting and expressed as disintegrations per minute (dpm) per lymph node for each experimental group. Stimulation Indices of 3, 3, 8, 17.6 and 25.2 were observed at concentrations of 2.5, 5, 10, 25 and 50% respectively. The EC3 was calculated to be <2.5% and no NOEC could be derived. Based on these results the substance is considered to be a skin sensitiser.

Constituent C&L information

As from the results of the available LLNA for Basil oil it is not possible to conclude on the skin sensitisation category, classification information for the constituents of this UVCB was taken into account. The major constituent linalool (range 45-62%) is classified as Skin Sensitiser 1B based on an EC3 value of 35.5% (available in the disseminated REACH dossier, accessed in April 2018). Also for other major constituents the EC3 value is at least above 2%, which is the classification limit for differentiation between Skin Sens. 1A and 1B in the CLP Regulation. In addition, the reported C&L for the remaining constituents does not indicate any concern for Skin Sens. 1A classification (ECHA disseminated dossiers and/or C&L notifications). Based on this information, a Skin Sens. 1B classification is likely to be appropriate for the UVCB as a whole.

Justification for classification or non-classification

Based on the available information for the UVCB as such and the classification of its constituents, the substance should be classified for skin sensitisation (Skin Sens. 1B / H317) in accordance with the criteria outlined in the EU CLP Regulation (1272/2008/EC and its amendments).