Silybum marianum, ext.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD guideline 429 ”Skin Sensitisation: Local Lymph Node Assay” adopted on 22 July 2010.

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

Silybum marianum, ext.

EC Number:

283-298-7

EC Name:

Silybum marianum, ext.

Cas Number:

84604-20-6

Molecular formula:

Not available - UVCB substance

IUPAC Name:

Extract of Chardon marie without support

Test material form:

liquid

Details on test material:

Name: Extract of Chardon marie without support
Batch/Lot number: VDO06205A
Appearance: Light yellow brown cloudy oil
Purity: Considered as 100%
Retest date: 13 July 2019
Storage conditions: Refrigerated
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.


Name: Extract of Chardon marie without support
Batch/Lot number: ABT01059A
Appearance: Brown/orange turbid oil
Purity: Considered as 100%
Retest date: 19 July 2018
Storage conditions: Refrigerated
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Reference: MPB-693061 / MPF-00024688
Batch number: VDO06126E
Provider: Pierre Fabre
Reference: GD 1508
Certificate of analysis: Yes
MSDS: Yes
Type: Raw material
Form: Liquid
Nature: Complex mixture
Colour: Yellowish
Storage conditions: Stored at room temperature
Requested test conditions: To be tested at 100 % (as is)
Sent quantity: 10 g
Expiry / Retest date: 27 November 2016

Specific details on test material used for the study:

No further details specified in the study report.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

The study is performed in 30 brown female mice SPF CBA/Ca (CBA/CaOlaHsd) from Envigo Nederland, NL-5961 NM Horst, Holland. Six (6) animals are included in the sighting study and 24 animals are included in the main study. At start of the acclimatisation period, the mice are 7-8 weeks of age and their body weight is within ± 20% of the mean body weight. In addition, 2 extra animals will be available until the second day of the main study for replacement purposes.
A pre-treatment period of at least 5 days (including an acclimatisation period of 5 days) is allowed during which the animals are observed daily in order to reject animals in poor condition or at the extremes of the weight range. All observations are recorded.

Housing
The study took place in an animal room provided with filtered air at a temperature of 21 ± 3°C and relative humidity of 55 ± 15%. The temperature and relative humidity in the animal room is recorded hourly during the study and the records will be retained.
The ventilation system has been designed to give 10 air changes per hour. The room will be illuminated to give a cycle of 12 hours light and 12 hours darkness. Light will be on from 06:00 h to 18:00 h.
The animals are group-housed and kept in transparent polycarbonate (macrolone type III) cages (floor area: 810 cm2).

Bedding
Bedding is made of Shepherd Specialty paper (“Alpha Dri” from Brogaarden, Soebredden 27, 2820 Gentofte). Analyses for relevant possible contaminants are performed regularly. Certificates of analysis are retained.

Environmental enrichment
Each cage contains a mouse iglooTM (Bio-serv). The mouse igloo allows the animals to show a wide range of natural behaviours. Certificates of analysis are retained.

Diet
A complete pelleted rodent diet "Altromin 1314 Fortified" (for growing animals) is available ad libitum. Altromin has been supplied by Altromin Spezialfutter GmbH & Co. KG, D-32791 Lage, Germany. Analyses for major nutritive components and relevant possible contaminants are performed regularly. Certificates of analysis are retained.

Drinking water
The animals have free access to bottles with domestic quality drinking water. Analyses for relevant possible contaminants are performed regularly on the drinking water. Certificates of analysis are retained.

Animal randomisation and allocation
On the day of arrival, the animals are allocated randomly to 9 groups (3 sighting groups of 2 animals and 6 main study groups of 4 animals) and a group of extra animals, using a randomisation scheme.
Prior to commencement of treatment, the animals in the main study may be re-allocated in order to reduce possible inter-group mean body weight differences. Data available from pre-treatment observations and clinical signs will be taken into account when re-allocating animals. If it is observed that an animal has skin irritation/lesion on the ears on the day before start of dosing, the animal will be excluded from the study and will be replaced by an extra animal.
No later than the second day of the main study, the extra animals will be killed or allocated to Citoxlab Denmark stock, after which they will no longer be part of this study.

Animal and cage identification
On arrival, each animal will have a chip implanted subcutaneously in the neck region identifying the animal. Each cage is identified by a colour coded card containing at least study number, group number, sex and animal number.

Study design: in vivo (LLNA)

Vehicle:

unchanged (no vehicle)

Concentration:

50%, 75% & 100%

No. of animals per dose:

Sighting study: 2 animals/ dose group
Main study: 4 animals/dose group

Details on study design:

Sighting study
Each animal is topically dosed with 25 µL of the appropriate formulation, using a pipette, on the dorsal surface of both ears (total dosing volume is 50 µL/animal).
The sighting study animals are dosed daily on three consecutive days. In order to prevent the animals from grooming the application sites after treatment, each mouse is restrained for minimum 30 sec. before being returned to the cage.
The animals in Group 1 and 2 were dosed on Day 1-3 and terminated on Day 6, and the animals in Group 3 were dosed on Day 3-5 and terminated on Day 8.

Main study
Each animal was topically dosed with 25 µL of the appropriate formulation, using a pipette, on the dorsal surface of both ears (total dosing volume is 50 µL/animal).
The main study animals are dosed daily on three consecutive days (Days 10-12). In order to prevent the animals from grooming the application sites after treatment, each mouse is restrained for minimum 30 sec. before being returned to the cage.
On Day 15 at 72 ± 2 hours after the last treatment on Day 12, each animal is injected intravenously in the tail vein with 250 µL of “3HTdR solution, 80 µCi/mL” per animal.

Clinical signs
All visible signs of ill health and any behavioural changes are recorded daily. Any deviation from normal is recorded.
In addition it is recorded if any animal shows mortality/morbidity at the start and end of the day.
Clinical signs observed during the pre-treatment period are only reported if considered relevant for the study. However, all findings will be listed in the raw data.

Skin reactions
On the day of re-allocation (main study animals), and from the first day of dosing (pre-dose) and until termination of the animals the application sites (ears) of the sighting and main study animals are observed for skin reactions. Any dermal irritation is scored according to the OECD Guideline for Testing of Chemicals No. 404, adopted 28th July, 2015: "Acute Dermal Irritation/Corrosion".

Ear thickness measurements – sighting study
Ear thickness measurements are taken using a thickness gauge at pre-dose (Day 1 for Groups 1 and 2, and Day 3 for Group 3), approximately 48 hours after the first dose (Day 3 for Groups 1 and 2, and Day 5 for Group 3), and just prior to termination (Day 6 for Groups 1 and 2, and Day 8 for Group 3).

Mortality
If an animal dies or is euthanized for ethical reasons after start of treatment, the animal will be necropsied and subjected to the procedures described in the section Terminal observations.

Body weight
Individual body weights are recorded at least once weekly, including the day of arrival, the day of re-allocation (main study animals), the first day of dosing and at termination. Body weight gain will be calculated.

Terminal observations
The animals were placed in a chamber with atmospheric air upon which a mixture of 70% CO2 and 30% O2 is applied at a steadily increasing concentration of CO2 for anaesthesia. The animals are monitored closely while in the chamber. Hereafter the animals are euthanized by cervical dislocation followed by exsanguination. On day 6 in the main study (Day 15), this will be done five hours (± 45 minutes) after dosing with “3HTdR solution, 80 µCi/mL”.
Ear thickness of the sighting study animals are determined by ear punch weight determinations. From each sighting animal, a 8 mm punch skin biopsy will be taken from each ear. The biopsies will be taken as close as possible to the tip of the ears. The biopsi samples from each animal will be weighed together. No further examinations of the sighting animals will be performed.

Proliferation assay – main study animals
Removal and preparation of auricular lymph nodes
The draining auricular lymph nodes are excised by making an incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the relevant lymph nodes. The auricular lymph nodes are carefully removed using forceps, and the lymph nodes from each animal are weighed together. The lymph nodes from each group will be pooled and placed into 2 mL PBS solution cold (+2-8ºC) in a single use container. The carcasses will be discarded after removal of the lymph nodes.

Preparation of single cell suspensions of lymph node cells
The following procedure are performed for each group of pooled lymph nodes:
Together with the 2 mL PBS solution, the lymph nodes were transferred to a single use 100 µm nylon cell strainer (BD Biosciences) placed in a 50 mL disposable plastic tube. By using the plunger of a disposable 2 mL syringe, the lymph nodes are disaggregated through the cell strainer. The mesh is washed with 8 mL cold PBS solution, collecting the cells in a total volume of approximately 10 mL PBS solution. Cell suspension is transferred to a 15 mL transparent tube with “conic” bottom and pelleted with a relative centrifugal force of 440 G for 10 minutes at room temperature.
After centrifugation, the supernatant is discarded and the remaining pellet is gently agitated before being re-suspended in cold PBS solution for a total volume of approximately 10 mL.
The washing step will be repeated, for a total of three washes.

Precipitation of macromolecules with TCA
Each of the pellet tubes are agitated gently to obtain a homogeneous cell suspension. For precipitation of the macromolecules, the cell suspension is sucked up into a disposable single-use pipette and dispensed into 3 mL 5% (w/v) TCA in purified water by placing the tip of the pipette under the fluid surface prior to slowly injecting the cell suspension into the fluid. The suspension is repeatedly be sucked up again in the pipette and afterwards returned until no large concentration of residual cells is observed in the tip of the pipette.
The tubes containing the cell suspensions are incubated for 18 hours ± 1 hour at +2-8ºC.

Determination of incorporation of 3HTdR (Day 7 in the main study; study Day 16)
After incubation for 18 hours ± 1 hour, the precipitates from the cell suspensions are recovered by centrifugation at 440 G for 10 minutes at room temperature. The supernatant is discarded. The pellet is agitated and the suspension is sucked up in a single-use pipette and dispensed into the same tube containing 1 mL 5% (w/v) TCA in purified water by placing the tip of the pipette under the fluid surface prior to slowly injecting the suspension in the fluid. The suspension is repeatedly be sucked up again in the pipette and afterwards returned until no large concentration of precipitates is observed in the tip of the pipette. The suspensions are dispersed using an ultrasonic lukewarm water bath for 5 minutes and are transferred to a 20 mL scintillation vial containing 15 mL of scintillation liquid. The vials are thoroughly agitated.
For measurement of the background, a sample containing 1 mL 5% (w/v) TCA in purified water in 15 mL scintillation liquid was prepared. Furthermore, a sample containing 10 µL of a 10 times diluted “80 µCi/ml (v:v) 3HTdR solution” in 15 mL scintillation liquid was be prepared either on Days 15 or 16.
The samples containing the scintillation liquid are incubated in darkness for at least 1 hour prior to measuring 3HTdR in the scintillation counter.
The b-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Positive control substance(s):

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

Statistics:

Data is processed to give group mean values and standard deviations where appropriate.

Results and discussion

Positive control results:

The SI for the positive control is 12.3 confirming the result of the test.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

7.1

Test group / Remarks:

50% formulation

Results 2

Key result

Parameter:

SI

Value:

6.8

Test group / Remarks:

75% formulation

Results 3

Key result

Parameter:

SI

Value:

4.3

Test group / Remarks:

100% formulation

Cellular proliferation data / Observations:

Note that no dermal scores were seen, thus the SI does not reflect irritation.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In order for a compound to be evaluated as positive the results of the stimulation index (SI) should be above 3.
Looking at the results of both the 50%, 75% and 100% formulations these are also found to be positive.
The 50%: SI = 7.1
The 75%: SI = 6.8
The 100%: SI= 4.3
Note that no dermal scores were seen, thus the SI does not reflect irritation.

Executive summary:

The objective of the study is to determine the skin sensitisation potential of Extract of Chardon marie without support.

The methods used in this study was be based on the OECD guideline 429 ”Skin Sensitisation: Local Lymph Node Assay” adopted on 22 July 2010. The CBA/Ca mouse has been selected as the test model, according to this guideline.

Prior to the conduction of the main study (LLNA) a sighting study (irritation/toxicity screen) was be performed, to determine the highest dose concentration tolerated in the animals.

The maximum dose for the sighting study was determined based on the OECD guideline 429 which recommends dosage at 100%, if possible. The low dose was set at 25%.

 

In order for a compound to be evaluated as positive the results of the stimulation index (SI) should be above 3.

The SI for the positive control is 12.3 confirming the result of the test.

 

Looking at the results of both the 50%, 75% and 100% formulations these are also found to be positive.

The 50%: SI  = 7.1

The 75%: SI = 6.8

The 100%: SI= 4.3

Note that no dermal scores were seen, thus the SI does not reflect irritation.