Saccharomyces cerevisiae cell wall, extracted

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 09 June 2011 to 19 december 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

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 cell wall, extracted

EC Number:

949-711-6

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Saccharomyces cerevisiae cell wall, extracted

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier (F-53941 Le Genest Saint Isle)
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: not specified
- Age at study initiation: 8 or 9 weeks old
- Weight at study initiation: 19.5 to 22.4 g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes. Animals were provided with enviromnental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Diet (e.g. ad libitum): food (M20, SDS) ad libitum
- Water (e.g. ad libitum): tap water from public distribution system ad libitum
Microbiological and chemical analyses of the water were carried out once every six months by IPL, Sante, Environnement Durables -Atlantique (Bordeaux).
- Acclimation period: at least 5 days
- Indication of any skin lesions: prior to the start of treatment, all animals are examined to ensure that they have no observable skin lesions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12 (continuous light from 07.00 to 19.00).
- IN-LIFE DATES: not specified

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0, 10, 25 and 50% (w/w) of the test item diluted in Acetone/Olive Oil (4:1 v/v) (AOO).

No. of animals per dose:

4 mice/dose (an additional mouse was treated in each group in case of problem which may occur during the study, in particular during the excision of lymph nodes. As no problem occurred, the lymph nodes of the additional mouse were not collected and only the data concerning the 4 first animals of each group were used in the study.

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The test item is prepared in a suitable vehicle at the required concentrations. The vehicle should be selected on the basis of maximising the test concentrations and solubility whilist producing a solution/suspension suitable for applicatio, of the test item. In order of preference, recommended vehicles are acetone/olive oil (4:1 v/v), dimethylformamide, methyl ethyl ketone, propylene glycol and dimethyl sulphoxide. According to tehe results, acetone/olive oil (4:1 v/v) (AOO) was determined to be the most suitable vehicle (brown solution) as it produced the most suitable formulation at the required concentration.
- Irritation / Systemic toxicity/ Ear thickness measurements / Eryhthema scores: as no available information was available regarding irritant potential or systemic toxicity of the test item in the mouse, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test item diluted at 50% in AOO to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed daily from day 1. Any signs of toxicity or excessive local irritation noted during this period were recorded. Ear thickness was recorded on day 1, day 3 and on day 6. The bodyweight of the mouse was recorded on Day 1 (prior to dosing) and Day 6.

POSITIVE CONTROL
- A study (29 december 2010 to 03 January 2011) was performed to assess the sensitivity of the strain of mouse used to a known sensitiser (a-Hexylcinnamaldehyde; CAS No 101-86-0). Three groups, each of four animals, were treated with 50 µL (25µL per ear) of a-Hexylcinnamaldehyde as a solution in AOO at concnetrations of 5, 10 and 25% (v/v). A further control group of four animals was treated with AOO alone.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response: According to the OECD guideline, evaluation of LLNA results is based on the measurement of 3H­ thymidine incorporation into the lymph node cells in the first place, it is state that "other endpoints for assessment of proliferation may be employed provided there is justification and appropriate scientific support". Lymph node cell count is more direct measurement of cell proliferation than determination of DNA synthetis and therefore considered an appropriate parameter for evaluation of cell proliferation in the assay.
For cell count indicates such cut-off values are much lower, 1.4 times increase of stimulation index. This is understandable by the facts that cell count indices have:
--> lower individual variance compared to 3H-thymidine incorporation
--> lower maximum stimulation indices compared to radioactive labelling
The internal validations of the cut-off value 1.4 instead of 3 was presented in the study report.
The difference of the cut-off values by 3H-thymidine incorporation and by cell counting were published by Bayer Healthcare on 20 June 2007 (data package 1 & data package 2) and on 28 June 2007 (data package 3). The value 1.5 was selected if the test was carried out with balb/c mice (Ebling Get al. (2005) Toxicology, 212, 69-79).

The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 1.4 compared to control values.
Other relevant criteria such as dose-response and irritation level were also taken into account for the interpretation of the results.
Any test item failing to produce a SI < 1.4 will be classified as a "non sensitiser".


TREATMENT PREPARATION AND ADMINISTRATION:
- Groups of four mice were treated with the test item diluted at concentrations of 50% (w/w), 25% (v/v), and 10% (v/v) in the vehicle AOO.
- The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).
- The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
- A further group of four mice received the vehicle alone in the same manner.
- An additional mouse was treated in each group in case of problem which may occur during the study, in particular during the excision of lymph nodes. As no problem occurred, the lymph nodes of the additional mouse were not collected and only the data concerning the 4 first animals of each group were used in the study.

CLINICAL OBSERVATIONS AND MORTALITY
-All animals were observed daily on Days 1, 2, 3, 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.

BODY WEIGHTS
- The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

EAR THICKNESS MEASUREMENTS AND RECORDIN OF LOCAL REACTIONS
- Ear thickness measurements and recording of local reactions were perfonned in order to assess any possible irritant effect of the test item, as possible irritancy may be involved in false positive lymphoproliferative responses.
- On day I and on day 3 (before application) as well as on day 6 (after sacrifice) of each experiment, the thickness of the right ear of each animal of the vehicle control and treated groups was measured by a micrometer.
- Furthermore, on day 6, punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and weighed in order to assess to the irritation potential of the test item and the two lymph nodes per mouse were weighed.
- Any irritation reaction (erythema and oedema) was recorded in parallel.
- Any other observation (dryness, presence of residual test item...) was noted.
- The irritation level of the test item is determined according to the following:
--> % increase in ear thickness between day 1 and day 3 and/or between day 1 and day 6 < 10%: Non irritant
--> % increase in ear thickness between day 1 and day 3 and/or between day 1 and day 6 = 10-25 %: Slightly irritant*
--> % increase in ear thickness between day 1 and day 3 and/or between day 1 and day 6 > 25 %: Irritant
--> The test item should also be considered as an irritant if the score of erythema is higher or equal to 3.
* while statistically significant increases can occur when ear thickness is less than 25% they have not been associated specifically with excessive irritation, expect if an increase of skin biopsy weight is noted (i.e. >25%).

TERMINAL PROCEDURE

Termination: On day 6 (end of the test), the animals were anaesthetised with sodium pentobarbital and administration continued to fatal levels. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group.

Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells of 4 mice of each group was prepared by gentle mechanical tissue disaggregation through a 200-mesh cell strainers in 4 mL of PBS (Ca2+/Mg2- free) containing 0.5% BSA into a well of a multi well 6. 10µL of this cell suspension was diluted in 10 mL of physiological saline solution (NaCl 0.9%). The lymphocyte cells were counted using a cell counter (Beckman Coulter Z2).
For the run, the lower size selected was 5 µm and the upper size selected was 15 µm (the average size of a lymphocyte is 8 µm).

STIMULATION INDEX DETERMINATION
- The proliferation response of lymph node cells was expressed as the number of lymphocytes per lymph node and as the ratio of lymphocytes into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
- Results are expressed as the Stimulation Index (SI).
- When using the pooled approach, the SI is calculated according to the following formula:
SI = Cell count of treated group/ cell count of control group

DETERMINATION OF THE EC1.4 VALUE
- The EC1.4 value (theoretical concentration resulting in a SI value of 1.4) was determined by linear interpolation of points on the dose-response curve, immediately above and below the 1.4-fold threshold. The equation used for calculation of EC1.4 was:
EC1.4 = c + [(1.4 - d) / (b - d)] x (a - c)
Legend:
a = the lowest concentration giving stimulation index > 1.4
b = the actual stimulation index caused by a
c = the highest concentration failing to produce a stimulation index of 1.4
d = the actual stimulation index caused by c
For cell count indicates such cut-off values are much lower,1.4 times increase of stimulation index. This is understandable by the facts that cell count indices have:
--> lower individual variance compared to 3M-thymidine incorporation
--> lower maximum stimulation indices compared to radioactive labelling

Positive control substance(s):

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

Results and discussion

Positive control results:

The Stimulation Index expressed as the cell count for each treatment group divided by the mean cell count of the vehicle control group are as follows:
- 5%: 1.21
- 10%: 1.47
- 25%: 2.41
The EC1.4 was calculated to be equal to 8.65%. The substance is thus correctly identfied as a skin sensitiser.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

Test item at 10%

Results 2

Key result

Parameter:

SI

Value:

0.97

Test group / Remarks:

Test item at 25%

Results 3

Key result

Parameter:

SI

Value:

0.84

Test group / Remarks:

Test item at 50%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA :
The cell counts (x106 cells/mL) per group were as follows:
- Control (AOO): 23.69
- Test item at 10%: 30.89
- Test item at 25%: 23.05
- Test item at 50%: 19.85

DETAILS ON STIMULATION INDEX CALCULATION
- No stimulation index of more than 1.4 was recorded in the study for the three concentration of the test item.
- The Stimulation Index (SI) calculated by pooled approach was 1.30, 0.97 and 0.84 for the treated groups at 10%, 25% and 50%, respectively.

EC1.4 CALCULATION
- The EC1.4 cannot be determined in this study.

CLINICAL OBSERVATIONS
- No mortality and no signs of systemic toxicity were noted in the test and controls animals during the test.

BODY WEIGHTS
- Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

LOCAL IRRITATION
- No cutaneous reaction was noted in the treated animals.
- No significant increase in ear thickness and in ear weight was noted in animals treated at 10%, 25% and 50%. Therefore, the test item must be considered as not excessively irritant at the three concentrations.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item Saccharomyces cerevisiae cell wall, extracted did not show a skin sensitisation potential in the murine Local Lymph Node Assay. Thus, the test item is not classified as a skin senstiser according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (UN-GHS) criteria.

Executive summary:

This GLP-compliant study was performed to assess the skin sensitisation potential of Saccharomyces cerevisiae cell wall, extracted, according to OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay) and EU method B.42 (Skin Sensitisation: Local Lymph Node Assay).

Material and methods

A preliminary irritation test was first performed in order to define the concentrations of test item to be used in the main test.

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 Acetone: Olive oil 4:1 (v/v) mixture (AOO). The 50 % (w/v) formulation was the highest concentration which was suitable for the test. 50% (w/v) formulation appeared to be a brown solution by visual examination.

The Preliminary Irritation/Toxicity Tests were performed in CBA/J mice using the single dose of 50% in AOO (using a single animal). Based on the observations recorded in the preliminary tests, 50 % (w/v) was selected as top dose for the main test.

In the main assay, sixteen female CBA/J mice were allocated to four groups of four animals each:

- three groups received Saccharomyces cerevisiae cell wall, extracted (formulated in AOO) at 10, 25 and 50 % (w/v) concentrations respectively,

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

The 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. Starting from Day 6, the cell proliferation in the local lymph nodes was assessed by counting the lymphocytes cells using a cell counter and the values obtained were used to calculate stimulation indices (SI) in comparison with the negative control group.

A study (29 December 2010 to 03 January 2011) was performed to assess the sensitivity of the strain of mouse used to a known sensitiser (α-Hexylcinnamaldehyde; CAS No 101-86-0). Three groups, each of four animals, were treated with 50 µL (25µL per ear) of α-Hexylcinnamaldehyde as a solution in AOO at concentrations of 5, 10 and 25% (v/v). A further control group of four animals was treated with AOO alone.

Results

No mortality and no signs of systemic toxicity were noted in the test and controls animals during the test.

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

No cutaneous reaction was noted in the treated animals. No significant increase in ear thickness and in ear weight was noted in animals treated at 10%, 25% and 50%. Therefore, the test item was not considered to be excessively irritant at the three concentrations.

The stimulation index values were 1.30, 0.97 and 0.84 at concentrations of 10, 25 and 50% (w/v), respectively. No stimulation index of more than 1.4 was recorded in the study for the three concentration of the test item.

The results of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle gave a lymphoproliferative response in line with historical positive control data; this result confirmed the validity of the assay.

Conclusion

Under the experimental conditions of this study, the test item Saccharomyces cerevisiae cell wall, extracted did not show a skin sensitisation potential in the murine Local Lymph Node Assay. Thus, the test item is not classified as a skin senstiser according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (UN-GHS) criteria.