Calcium tartrate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 02 December 2010 to 28 December 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (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 tartrate

EC Number:

221-621-5

EC Name:

Calcium tartrate

Cas Number:

3164-34-9

Molecular formula:

C4H6O6.Ca

IUPAC Name:

Calcium carbonate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Name of test material (as cited in study report): TARTRATE DE CALCIUM
- Physical state: amber powder
- Analytical purity: 93.44%
- Lot/batch No.: CAMP 09-10
- Expiration date of the lot/batch: December 2011
- Storage conditions of test material: at room temperature, protected from light and humidity.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: on the first day of the treatment period, the animals of the preliminary test were approximately 10 weeks old and the animals of the main test were approximately 9 weeks old
- Weight at study initiation: a mean body weight of 20.0 g +- 1.3 g
- Housing: housed individually in disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm)
- Diet (e.g. ad libitum): free access to SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): tap water (filtered using a 0.22 micron filter)
- Acclimation period: the animals were acclimated to the study conditions for at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00 - 19:00).

IN-LIFE DATES: From: 02 December 2010 To: 28 December 2010.

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

0.5, 1, 2.5, 5 and 10%.

No. of animals per dose:

4 animals per dose.

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: due to the unsatisfactory solubility of the test item in the first recommended vehicle (acetone/olive oil (4/1, v/v)),
dimethylformamide, methyl ethyl ketone, dimethylsulfoxide and 1% pluronic L92, propylene glycol was chosen as vehicle. A homogeneous
suspension was obtained under agitation at the maximum concentration of 10%.
- Irritation: no cutaneous reactions and no notable increase in ear thickness were observed at any of the tested concentrations.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine Local Lymph Node Assay (LLNA) based on the design adopted by ICCVAM (Interagency Coordination Committee on
the Validation of Alternative Methods, ICCVAM 1999) and ECETOC (Monograph No. 78 Skin sensitization Testing for the Purpose of Hazard
Identification and Risk Assessment, September 2000), with the addition of the evaluation of local irritation.
- Criteria used to consider a positive response: stimulation Indice SI >= 3.

TREATMENT PREPARATION AND ADMINISTRATION:
- Treatment preparation: the concentrations were expressed in % (w/v).
The test item was prepared at the chosen concentrations in PG. The dosage form preparations were homogenized by magnetic agitation.
The reference item was dissolved in AOO at the concentration of 25% (v/v).
- Administration: on days 1, 2 and 3, a dose-volume of 25 µL of the control or dosage form preparations was applied to the dorsal surface of both
ears, using an adjustable pipette fitted with a plastic tip.
In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration.
No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each
application.

Positive control substance(s):

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

Results and discussion

Positive control results:

The mean cell viability in the vehicle group was higher than 70% and the threshold positive value of 3 for the SI was reached in the positive control
group. The study was therefore considered valid.

In vivo (LLNA)

Results

Results 1

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

DPM per group: Group 1: Vehicle: 504.00 Group 2: 0.5%: 488.00 Group 3: 1%: 355.00 Group 4: 2.5%: 701.00 Group 5: 5%: 330.00 Group 6: 10%: 424.00 DPM per node: Group 1: Vehicle: 63.00 Group 2: 0.5%: 61.00 Group 3: 1%: 44.38 Group 4: 2.5%: 87.63 Group 5: 5%: 41.25 Group 6: 10%: 53.00

Results 2

Key result

Parameter:

SI

Value:

0.97

Test group / Remarks:

Group 2: 0.5%:

Results 3

Key result

Parameter:

SI

Value:

0.7

Test group / Remarks:

Group 3: 1%

Results 4

Key result

Parameter:

SI

Value:

1.39

Test group / Remarks:

Group 4: 2.5%

Results 5

Key result

Parameter:

SI

Value:

0.65

Test group / Remarks:

Group 5: 5%

Results 6

Key result

Parameter:

SI

Value:

0.84

Test group / Remarks:

Group 6: 10%

Any other information on results incl. tables

Clinical signs and mortality

Neither mortality nor clinical signs were observed during the study.

Body weight

The body weight change of treated animals was similar to that of control animals.

Local irritation

No cutaneous reactions and no notable increase in ear thickness were observed at any of the tested concentrations.

Proliferation assay

The mean cell viability in the vehicle group was higher than 70% and the threshold positive value of 3 for the SI was reached in the positive control group. The study was therefore considered valid.

No notable lymphoproliferation was noted with the test item at any tested concentrations.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test item, TARTRATE DE CALCIUM, did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.

Executive summary:

The objective of this study was to evaluate the potential of the test item, TARTRATE DE CALCIUM, to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA). Evaluation of local irritation was also carried out in parallel.

This study was conducted in compliance with the principles of Good Laboratory Practice.

 

Methods

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

In the main test, 28 female CBA/J mice were allocated to 7 groups:

.           five treated groups of four animals receiving the test item at the concentration of 0.5, 1, 2.5, 5 or 10% in propylene glycol (vehicle),

.           one negative control group of four animals receiving the vehicle,

.           one positive control group of four animals receiving the reference item, alpha-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25% in a mixture acetone/olive oil (4/1; v/v).

 

During the induction phase, the test item, vehicle or reference item was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate Stimulation Indices (SI).

 

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

 

Results

Following the solubility assay, propylene glycol was chosen as vehicle. A homogeneous suspension was obtained under agitation at the maximum concentration of 10%.

Consequently, the concentrations selected for the preliminary test were 1, 2.5, 5 and 10%.

Since the test item was non-irritant in the preliminary test, the highest concentration retained for the main test was the maximal practicable concentration (10%).

Neither mortality nor clinical signs were observed during the study.

The body weight change of treated animals was similar to that of control animals.

No cutaneous reactions and no notable increase in ear thickness were observed at any of the tested concentrations.

As all acceptance criteria were met, this experiment was therefore considered valid.

The results are presented in the following table:

Treatment	Concentration (%)	Irritation level	Stimulation Index (SI)
Test item	0.5	non-irritant	0.97
Test item	1	non-irritant	0.70
Test item	2.5	non-irritant	1.39
Test item	5	non-irritant	0.65
Test item	10	non-irritant	0.84
HCA	25	-	12.40

No notable lymphoproliferation was noted with the test item at any tested concentrations.

 

Conclusion

Under the experimental conditions of this study, the test item, TARTRATE DE CALCIUM, did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.