Fir, Abies balsamea, ext.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 January 2018 - 28 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

in accordance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

22 July 2010

Deviations:

yes

Remarks:

Minor deviation on the Temperature and Humidity range of the husbandry

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

6 July 2012

Deviations:

yes

Remarks:

See OECD TG 429

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Fir, Abies balsamea, ext.

EC Number:

285-364-0

EC Name:

Fir, Abies balsamea, ext.

Cas Number:

85085-34-3

IUPAC Name:

Fir Balsam Oil

Test material form:

liquid

Details on test material:

Name of test material as cited in study report: Fir Needle Oil Canadian (Fir, Abies Balsamea, ext)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Botanical source and Lot# 67919
- Expiration date of the lot/batch: 13 septembre 2018
- Purity test date: UVCB, considered 100% pure

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25 °C, ≤ 70 RH%), protected from light, avoid contact with iron.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was diluted in AOO (acetone:olive oil 4:1 (v:v) mixture) at the following concentration (w/v) 50%, 25%, 10%. An undiluted sample was also used in the Preliminary Compatibility Test.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd mice

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo, San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: not specified
- Age at study initiation: 9 weeks old (age-matched, within one week)
- Weight at study initiation: 19.2 - 21.7g (weight variation did not exceed +- 20 % of the mean weight)
- Housing: Mice were group caged in Type 2 polypropylene/polycarbonate cages with bedding from J. Rettenmaier & Söhne GmbH + Co.KG
- Diet (e.g. ad libitum): ad libitum (ssniff® SM Rat/Mouse by ssniff Spezialdiäten GmbH of Germany and Gel diet Transport by Scientific Animal Food & Engineering of France)
- Water (e.g. ad libitum): ad libitum (municipal water)
- Acclimation period: 14 days
- Indication of any skin lesions: none reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.4 - 27.4 C
- Humidity (%): 23 - 97%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12 hours light from 6am to 6pm

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Preliminary Compatibility Test: 50% (w/v) and 100% (undiluted)
Main study: 10%, 25%, 50% (w/v)

No. of animals per dose:

Preliminary Compatibility Test: 2
Main study: 4

Details on study design:

PRE-SCREEN TESTS: 2 animals/dose. Doses: 50% and 100%
- Compound solubility: 100% soluble in the vehicle (AOO)
- Irritation: None
- Systemic toxicity: None observed (no mortality). Body weight loss was within the acceptable range.
- Ear thickness measurements: Above acceptable limit for the 100% dose
- Erythema scores: Acceptable

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: The LLNA assesses proliferation in the draining auricular lymph nodes located in the cervical region at the bifurcation of the jugular vein. Lymphocyte proliferation in test groups is compared to that in the vehicle treated control. The ratio of the proliferation in test groups to that in the control, termed Stimulation Index (SI), is determined and must be at least equal or greater than three, for a test substance to be classified as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was found to be highly soluble in AOO (acetone:olive oil 4:1 (v:v) mixture) during a short Preliminary Compatibility Test. The formulations at 50% (w/v), 25%(w/v) and 10% (w/v) using AOO as vehicle were suitable for treatment. The formulations appeared to be solutions by visual examination.

During the study, animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Positive control substance(s):

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

Results and discussion

Positive control results:

No mortality was observed. Mean body weight of animals was in the acceptable range. DPN = 3662.7 and SI = 6.8 (both values are within the range of historical data). The results of the negative control were also within the range of historical data.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1

Test group / Remarks:

Negative Control

Results 2

Parameter:

SI

Value:

1.5

Test group / Remarks:

Concentration of test item: 10%

Results 3

Parameter:

SI

Value:

1.3

Test group / Remarks:

Concentration of test item: 25%

Results 4

Parameter:

SI

Value:

3.2

Test group / Remarks:

Concentration of test item: 50%

Results 5

Parameter:

EC3

Value:

47.4

Test group / Remarks:

Fir Needle Oil Canadian

Remarks on result:

other:

Remarks:

47.4% (w/v)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The cell proliferation in the local lymph nodes was assessed by measuring the incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. Where the DPN is DPM divided by the number of lymph nodes and DPM is the number of radioactive disintegrations per minute

EC3 CALCULATION
EC3 or EC3 means the effective chemical concentration required for SI=3 was calculated by linear interpolation according to the equation:
EC3 = c + [(3-d)/(b-d)] x (a-c)
where the data points lying immediately above and below the SI value of 3 on the LLNA dose-response plot have the co-ordinates (a,b) and (c,d) respectively [9,10].

CLINICAL OBSERVATIONS:
No mortality or systemic toxicity was observed during the main study. No test item precipitate was observed on the ears of the experimental animals. There were no indications of any irritancy at the site of application (See attached full report for details).

BODY WEIGHTS
Marked body weight loss (≥5%) was observed for one animal of the 50% (w/v) dose group and for one animal of the positive control group. However, the mean body weight of these groups were in the acceptable range and these changes were considered as individual variability. No treatment related effects were observed on the mean body weight changes in the main study (See attached full report for details).

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Fir Needle Oil Canadian (Fir, Abies Balsamea, ext), tested in a suitable vehicle, was shown to have weak sensitization potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value of present Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) is 47.4% (w/v).

The following classification/labelling is triggered: Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 7) 2017: Category 1 (sub-category 1B).

Executive summary:

The aim of this study was to determine the skin sensitisation potential of Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) following dermal exposure in mice. The study was performed with vertebrate animals as no full regulatory in vitro alternative is available. The minimum number of animals was used, in accordance to the regulatory guidelines for animal welfare.

 

Based on the results of the Preliminary Compatibility Test, the test item characteristics, its usage and on the recommendations of the OECD Guideline [1], the best vehicle for the test item was AOO (acetone:olive oil 4:1 (v:v) mixture). The formulations at 100% (undiluted), 50% (w/v), 25% (w/v) and 10% (w/v) concentrations, using AOO as vehicle, were suitable for the test. The formulations appeared to be solutions by visual examination.

 

The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose) 100% (undiluted) and 50% (w/v) in AOO. Based on the observations recorded in the preliminary test, the 50% (w/v) dose was selected as top dose for the main test.

 

In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each:

-   three groups received Fir Needle Oil Canadian (formulated in AOO) at 50% (w/v), 25% (w/v) and 10% (w/v) concentrations,

-   the negative control group received the vehicle (AOO) only,

-   the positive control group received 25 % (w/v) HCA (dissolved in AOO).

 

The test item formulations were applied on the dorsal surface of ears of experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was assessed by measuring the incorporation of tritiated methyl thymidine (³HTdR) and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

 

No mortality or systemic toxicity was observed during the main study. No test item precipitate was observed on the ears of the experimental animals.There were no indications of any irritancy at the site of application. No treatment related effects were observed on the mean body weights in the main study.

 

The stimulation index values were 3.2, 1.3 and 1.5 at concentrations of 50% (w/v), 25% (w/v) and 10% (w/v), respectively.

 

The result of the positive control substance (a-Hexylcinnamaldehyde, HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay [1]. A lymphoproliferative response (SI = 6.8) in harmony with historical control data was noted for the positive control chemical, this result confirmed the validity of the assay.

 

In conclusion, under the conditions of the present assay, Fir Needle Oil Canadian (Fir, Abies Balsamea, ext), tested in a suitable vehicle, was shown to have weak sensitization potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value of present Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) is 47.4% (w/v).

 

The following classification/labelling is triggered: Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 7) 2017: Category 1 (sub-category 1B).