Sulfates of potassium, sodium and calcium, byproduct from fermentation

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 10 April 2017 to 24 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

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:

calcium dipotassium disodium trisulfate

Molecular formula:

SO42-, K+, Na+, Ca2+

IUPAC Name:

calcium dipotassium disodium trisulfate

Test material form:

solid

Remarks:

light to beige solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: EXP LIberica Lote: AI16503047
- Expiration date of the lot/batch: 30 November 2019
- Purity test date: 1 February 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25ºC, below 70 RH%), protected from light and humidity.
- Stability under test conditions: not applicable
- Solubility and stability of the test substance in the solvent/vehicle:The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 100% (w/v). The formulations appeared to be suspensions by visual examination.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was weighed and formulations prepared daily on a weight:volume basis (as % (w/v))
FORM AS APPLIED IN THE TEST (if different from that of starting material) : in solution in propylene glycol

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

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 at arrival; standard housing conditions during the study
- Age at study initiation: 10 weeks old (age-matched, within one week)
- Weight at study initiation: 20.0 – 20.8 grams
- Housing: Type II. polypropylene / polycarbonate
- Diet (e.g. ad libitum): ssniff® SM Rat/Mouse – Breeding and Maintenance, 15 mm, autoclavable “Complete feed for Rats and Mice – Breeding and Maintenance" (Batch number: 484 14771) ad libitum
- Water (e.g. ad libitum): Animals received tap water from the municipal supply from 500 mL bottles, ad libitum
- Acclimation period: 21 days
- Indication of any skin lesions: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 – 25.9°C
- Humidity (%): 30 - 74 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
- IN-LIFE DATES: From: 10 May 2017 To: 5 June 2017

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

0, 25, 50 and 100%

No. of animals per dose:

4 animals were used per condition

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Based on the observation of the solubility test, the maximum available concentration was 100 % (w/v).
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
- 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.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was weighed and formulations prepared daily on a weight:volume basis (as % (w/v)).
Treatment with test item : 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.
Treatment with Tritiated thymidine : 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. The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. 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 in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C. After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

DETERMINATION OF INCORPORATED RADIOLABELLED THYMIDINE
After the final 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.

EVALUATION: 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.

Positive control substance(s):

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

Results and discussion

Positive control results:

The result of the positive control substance aplha-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25 % in the relevant vehicle (PG) using CBA/CaOlaHsd mice.

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 8.2) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.

Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were in within the historical control range. Each treated and control group included 4 animals.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

Sulfates of potassium, sodium and calcium, by-product from fermentation 100 % (w/v) in PG

Results 2

Key result

Parameter:

SI

Value:

0.6

Test group / Remarks:

Sulfates of potassium, sodium and calcium, by-product from fermentation 50% (w/v) in PG

Results 3

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

Sulfates of potassium, sodium and calcium, by-product from fermentation25% (w/v) in PG

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The stimulation index values were 0.8, 0.6 and 0.8 at concentrations of 100, 50 and 25 % (w/v), respectively.. The appearance of the lymph nodes was normal in the negative control group and in the 100, 50 and 25 % (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

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%) was observed on the mean body weight changes in any groups; however, the body weight loss was >=5% for 1/4 animals in the Positive 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#(%)
3999	1	Negative (vehicle) control	20.0	20.1	0.5
4005	2	in PG	20.8	22.2	6.7
3998	3		20.2	20.4	1.0
4004	4		20.3	19.8	-2.5
		Mean	20.3	20.6	1.4
4013	5	Sulfates of potassium, sodium and calcium, by-product from fermentation	20.1	19.8	-1.5
4006	6	100 % (w/v)	20.7	19.9	-3.9
4003	7	in PG	20.3	20.0	-1.5
4008	8		20.8	21.0	1.0
		Mean	20.5	20.2	-1.5
3996	9	Sulfates of potassium, sodium and calcium, by-product from fermentation	20.4	19.9	-2.5
4012	10	50 % (w/v)	20.4	21.2	3.9
4007	11	in PG	20.3	20.9	3.0
4002	12		20.1	19.2	-4.5
		Mean	20.3	20.3	0.0
4009	13	Sulfates of potassium, sodium and calcium, by-product from fermentation	20.4	20.8	2.0
3997	14	25 % (w/v)	20.5	19.7	-3.9
4011	15	in PG	20.3	19.5	-3.9
4001	16		20.6	20.4	-1.0
		Mean	20.5	20.1	-1.7
4015	17	Positive control	20.4	19.9	-2.5
4014	18	25 % (w/v) HCA	20.2	20.6	2.0
4010	19	in PG	20.8	20.6	-1.0
4000	20		20.0	18.9	-5.5
		Mean	20.4	20.0	-1.7

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)	34.5	-	-	-	-
Negative control (PG)	2017	1982.5	8	247.8	1.0
Sulfates of potassium, sodium and calcium, by-product from fermentation 100 % (w/v) in PG	1625	1590.5	8	198.8	0.8
Sulfates of potassium, sodium and calcium, by-product from fermentation 50 % (w/v) in PG	1246	1211.5	8	151.4	0.6
Sulfates of potassium, sodium and calcium, by-product from fermentation 25 % (w/v) in PG	1551	1516.5	8	189.6	0.8
Positive control (25 % (w/v) HCA in PG)	16300	16265.5	8	2033.2	8.2

 

Table 3:Individual Body Weights for all Animals with Group Means (Preliminary Irritation/Toxicity Test)

 

Animal	Identity Number 1 2	Test Group	Initial Body	Terminal Body	Change#
Number		Name	Weight (g)	Weight* (g)	(%)
3620		100 % (w/v)	18.9	19.1	1.1
3602		100 % (w/v)	19.2	18.5	-3.6
		Mean	19.1	18.8	-1.3
3607	3	50 % (w/v)	18.8	17.6	-6.4
3611	4	50 % (w/v)	19.9	18.5	-7.0
		Mean	19.4	18.1	-6.7

Notes:

1.         *: Terminal body weights were measuredonDay6.

2.         #: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x100

 

 Table 4:Individual Ear Thickness for all Animals (Preliminary Irritation/Toxicity Test)

 

Animal Number	Identity Number	Test Group Name	Ear Thickness on Day 1 (mm)		Ear Thickness on Day 3 (mm)		Ear Thickness on Day 6 (mm)		Biopsy weight* on Day 6 (mg)
			Right	Left	Right	Left	Right	Left
3620	1	100 % (w/v)	0.20	0.21	0.21	0.21	0.22	0.23	14.60
3602	2	100 % (w/v)	0.22	0.22	0.23	0.22	0.23	0.22	14.95
3607	3	50 % (w/v)	0.22	0.22	0.22	0.22	0.22	0.22	16.73
3611	4	50 % (w/v)	0.21	0.21	0.21	0.21	0.22	0.21	16.57

Note:

1.        *: Historical control range: 11.92-22.53 mg. Positive response isover28.16 mg(≥25%).

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, under the conditions of the present assay, Sulfates of potassium, sodium and calcium, by-product from fermentation, tested in a suitable vehicle, did not show a sensitisation potential (non-sensitizer) in the Local Lymph Node Assay. Hence the test item was not classified for sensitisation according to CLP regulation.

Executive summary:

The purpose of the GLP compliant study was to assess the potential sensitising effect of the test item Sulfates of potassium, sodium and calcium, by-product from fermentation when dermally applied on mice in a Local Lymph Node Assay performed accordingly to OECD 429 method.

The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 100 and 50 % (w/v) in Propylene Glycol. Based on the observations recorded in the preliminary test, 100 % (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 Sulfates of potassium, sodium and calcium, by-product from fermentation (formulated in PG) at 100, 50 and 25 % (w/v) concentrations respectively,

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

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

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 marked body weight losses (≥5%) was observed on the mean body weight changes in any groups; however, the body weight loss was ≥5% for 1/4 animals in the Positive control group. The stimulation index values  were  0.8,  0.6  and  0.8  at  concentrations  of  100,  50  and 25 % (w/v), respectively.

In conclusion, under the conditions of the present assay, Sulfates of potassium, sodium and calcium, by-product from fermentation, tested in a suitable vehicle, did not show a sensitisation potential (non-sensitizer) in the Local Lymph Node Assay. Hence the test item was not classified for sensitisation according to CLP regulation.