Saccharomyces cerevisiae, lysate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 6 September 2017 to 13 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Due to technical reasons, temperature values (maximum of 25.3°C) outside the expected ranges of 19-25°C and relative humidity values (minimum of 26% and maximum of 78%) outside the expected range of 30-70% were recorded occasionally during the study.

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:

Saccharomyces cerevisiae, lysate

EC Number:

305-230-8

EC Name:

Saccharomyces cerevisiae, lysate

Cas Number:

94350-12-6

IUPAC Name:

Saccharomyces cerevisiae, lysate

Test material form:

solid: particulate/powder

Remarks:

light beige

Details on test material:

- Source and lot/batch No.of test material:
supplied by the sponsor, batch no. AC17F00560
- Expiration date of the lot/batch: February 2019
- Purity test date: 30 June 2017

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AC17F00560
- Expiration date of the lot/batch: February 2019
- Purity test date: 30 June 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25°CC, ≤70 RH%**)
- Stability under test conditions: not applicable
- Solubility and stability of the test substance in the solvent/vehicle: soluble at the high dose

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was weighed and formulations prepared daily on a weight:volume basis (as % (w/v))

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd mice

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF (Specific Pathogen Free)
- Age at study initiation: 11 weeks old (age-matched, within one week)
- Weight at study initiation: 20.2 – 21.7 grams
- Housing: Type II. polypropylene / polycarbonate
- Diet (e.g. ad libitum): Animals received ssniff® SM Rat/Mouse – Breeding and Maintenance, “Complete feed for Rats and Mice – Breeding and Maintenance" and Gel diet Transport, ad libitum
- Water (e.g. ad libitum): tap water from the municipal supply from 500 mL bottles, ad libitum. - Acclimation period: 34 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 – 25.3°C
- Humidity (%): 26 - 78 % relative humidity
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/ 12 hours dark, light from 6.00 a.m. to 6.00 p.m.
- IN-LIFE DATES: From: 6 September 2017 To: 19 September 2017

Study design: in vivo (LLNA)

Vehicle:

other: 1 % Pluronic in distilled water

Concentration:

0, 5, 10, 25% in vehicle

No. of animals per dose:

4 animales were used per condition

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The solubility of the test item was examined in a short Preliminary Compatibility Test. The following standard OECD vehicles were assessed: Acetone: Olive oil 4:1 (v/v) mixture, N,N- dimethylformamide, Methyl ethyl ketone, Propylene glycol (1 % Pluronic), Dimethyl sulfoxide and 1 % aqueous Pluronic® PE9200. The best vehicle taking into account the test item characteristics, its usage and the requirements of the relevant OECD guideline was considered to be 1 % Pluronic. The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 25% (w/v). The formulations appeared to be suspensions by visual examination.

- Irritation: In the Preliminary Irritation / Toxicity Test, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored.
During the Preliminary Irritation / Toxicity Test, no mortality or signs of systemic toxicity were observed. No amount of test item precipitation was observed on the ears of the animals. There were no indications of any irritancy at the site of application

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Randomisation: The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups
- Criteria used to consider a positive response:
The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:

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.

On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (phosphate buffered saline) containing approximately 20 µCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide.

The draining auricular lymph nodes were excised. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels.

Once removed, the nodes of mice from each test group was pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.

A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected.After the washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5 % (w/v) TCA solution was added to the tubes for precipitation of macromolecules.

After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4oC), and supernatants were removed. Pellets were resuspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).

The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 % (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

Positive control substance(s):

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

Results and discussion

Positive control results:

No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historical positive control data (stimulation index value of 5.2) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Vehicle control

Results 2

Key result

Parameter:

SI

Value:

2.1

Test group / Remarks:

25% Test item

Results 3

Key result

Parameter:

SI

Value:

2.9

Test group / Remarks:

10% Test item

Results 4

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

5% Test item

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The appearance of the lymph nodes was normal in the negative control group and in the 25, 10 and 5 % (w/v) test item treated dose groups. Larger than normal lymph nodes were observed in the positive 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.

EC3 CALCULATION
No EC3 calculation was performed in this study.

CLINICAL OBSERVATIONS:
No mortality or signs of systemic toxicity were observed during the study. No amount of test item precipitation was observed on the ears of the animals.

BODY WEIGHTS
No marked body weight losses (≥5%) were observed on the mean body weight changes in any groups; however, the body weight loss was ≥5% for 1 out of 4 animals in the Negative control group. These changes were considered incidental

Any other information on results incl. tables

Table 1:Individual Body Weights for all Animals with Group Means

 

Animal Number	Identity Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight* (g)	Change^#(%)
4602	1	Negative (vehicle) control	21.5	19.8	-7.9
4606	2	in 1 %Pluronic	21.6	22.0	1.9
4612	3		20.3	20.4	0.5
4617	4		20.4	21.4	4.9
		Mean	21.0	20.9	-0.2
4603	5	Saccharomyces cerevisiae, lysate	21.2	21.9	3.3
4620	6	25% (w/v)	21.7	23.5	8.3
4618	7	in 1 %Pluronic	20.9	21.6	3.3
4607	8		20.4	21.5	5.4
		Mean	21.1	22.1	5.1
4616	9	Saccharomyces cerevisiae, lysate	21.3	22.7	6.6
4601	10	10 % (w/v)	21.7	22.1	1.8
4611	11	in 1 %Pluronic	20.7	22.2	7.2
4608	12		20.3	20.5	1.0
		Mean	21.0	21.9	4.2
4605	13	Saccharomyces cerevisiae, lysate	21.2	20.7	-2.4
4613	14	5 % (w/v)	21.5	21.2	-1.4
4615	15	in 1 %Pluronic	20.8	21.3	2.4
4609	16		20.5	21.6	5.4
		Mean	21.0	21.2	1.0
4610	17	Positive control	21.2	21.7	2.4
4614	18	25 % (w/v) HCA	21.7	21.4	-1.4
4619	19	in 1 %Pluronic	20.8	23.9	14.9
4604	20		20.2	21.0	4.0
		Mean	21.0	22.0	4.9

Notes:

*: Terminal body weights were measured on Day 6.

#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

Table 2:DPM, DPN and Stimulation Index Values for all Groups

 

Test Group Name	Measured DPM / group	DPM	Number of lymph nodes	DPN	Stimulation Index
Background (5 %(w/v)TCA)	34	-	-	-	-
Negative control (1 %Pluronic)	2999	2965.0	8	370.6	1.0
Saccharomyces cerevisiae, lysate 25 %(w/v)in 1 %Pluronic	6252	6218.0	8	777.3	2.1
Saccharomyces cerevisiae, lysate 10 %(w/v)in 1 %Pluronic	8608	8574.0	8	1071.8	2.9
Saccharomyces cerevisiae, lysate 5 % (w/v) in 1 %Pluronic	3922	3888.0	8	486.0	1.3
Positive control (25 % (w/v) HCA in 1 %Pluronic)	15382	15348.0	8	1918.5	5.2

The stimulation index values were 2.1, 2.9 and 1.3 at concentrations of 25, 10 and 5% (w/v), respectively.

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present assay, Saccharomyces cerevisiae, lysate, tested in a suitable vehicle, did not show a sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.
No classification/labelling is triggered according to CLP criteria.

Executive summary:

This GLP compliant study was performed according to OECD 429 guideline (Local Lymph Node Assay) in order to assess the potential sensitisation effect of the registered item, Saccharamyces Cerevisiae lysates, through skin application in mice.

Based on the results of the Preliminary Compatibility Test, the test item characteristics, its usage and on the recommendations of the OECD Guideline, the best vehicle for the test item was 1 % Pluronic. The 25 % (w/v) formulation was the highest concentration which was suitable for the test. The formulations appeared to be suspensions by visual examination.

The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 25 and 10 % (w/v) in 1 % Pluronic. Based on the observations recorded in the preliminary test, 25 % (w/v) 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 Saccharomyces cerevisiae, lysate (formulated in 1 % Pluronic) at 25, 10 and 5 % (w/v) concentrations respectively,

-       the negative control group received the vehicle (1 % Pluronic) only,

-       the positive control group received 25 % (w/v) HCA (dissolved in 1 % Pluronic).

The test item solutions 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 disintegrations per minutes after 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 signs of systemic toxicity were observed during the study. No amount of test item precipitation was observed on the ears of the animals. No marked body weight losses (≥5%) was observed on the mean body weight changes in any groups; however, the body weight loss was ≥5% for 1 out of 4 animals in the Negative control group.

The stimulation index values were 2.1, 2.9 and 1.3 at concentrations of 25, 10 and 5 % (w/v), respectively.

Under the conditions of the present assay, Saccharomyces cerevisiae, lysate, tested in a suitable vehicle, did not show a sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

No classification/labelling is triggered according to CLP criteria.