Calcium carbonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 January 2010 to 27 January 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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 carbonate

EC Number:

207-439-9

EC Name:

Calcium carbonate

Cas Number:

471-34-1

Molecular formula:

CH2O3.Ca

IUPAC Name:

calcium carbonate

Test material form:

solid: nanoform, no surface treatment

Details on test material:

- Name of test material (as cited in study report): Calcium carbonate (nano)
- Description: White powder
- Batch No.: GICM014427
- Storage condition of test material: Room temperature in the dark

The nano form of calcium carbonate was tested because this form was anticipated to represent the worst case as the smaller size particles are more likely to be transported through the skin.

Specific details on test material used for the study:

Uncoated nano calcium carbonate

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Bicester, Oxon, UK
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: individually in suspended solid-floor polypropylene cages
- Diet (e.g. ad libitum): 2014 Teklad Global Rodent diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: >= 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr):15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 13/01/2010 - 27/01/2010

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

5, 10 or 25 %w/w

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
Using available information regarding the systemic toxicity/irritancy potential of the test material, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µl of the test material at a concentration of 25 %w/w in dimethyl formamide to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6. No signs of systemic toxicity were noted. White staining on the ears was noted post dose on Days 2 and 3. Based on this information the dose levels selected for the main test were 25 %, 10 % and 5 %w/w in dimethyl formamide.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay in the Mouse. The assay has undergone extensive inter-laboratory validation and has been shown to reliably detect test materials that are moderate to strong sensitisers.
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ration of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test substance is regarded as a sensitiser if at least one concentration of the test substance results in a threefold or greater increase in 3HTdR incorporation compared to control values.

TREATMENT PREPARATION AND ADMINISTRATION: The test substance was freshly prepared as a suspension in dimethyl formamides. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. The test substance was formulated within 2 hours of it being applied to the test system. No analysis was conducted to determine the homogeneity, concentration or stability of the formulation. The mice were treated by daily application of 25 µL of the appropriate concentration of the test substance to the dorsal surface of each ear for 3 consecutive days. The test substance was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.

Positive control substance(s):

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

Results and discussion

Positive control results:

The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the control groups was 5.16, hence the result was positive.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.74

Test group / Remarks:

5 %w/w

Results 2

Parameter:

SI

Value:

1.13

Test group / Remarks:

10 %w/w

Results 3

Parameter:

SI

Value:

1.19

Test group / Remarks:

25 %w/w

Any other information on results incl. tables

Disintegrations per minute at each concentration

Concentration (%w/w in dimethyl  formamide)	dpm	dpm/node
Vehicle	8246.81	1030.85
5	14319.64	1789.96
10	9325.17	1165.65
25	15632.46	1954.06

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The substance was considered to be a non-sensitiser under the conditions of the test.

Executive summary:

A study was performed according to OECD Guideline 429 to assess the skin sensitisation potential of calcium carbonate (nano) in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. Three groups, each of four animals, were treated with 50 µl (25 µl per ear) of the test substance as a suspension in dimethyl formamide at concentrations of 5%, 10% or 25% w/w. A further group of four animals was treated with dimethyl formamide alone. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group was 1.74, 1.13 and 1.19 at concentrations of 5, 10 and 25 %w/w, respectively. The test material was considered to be a non sensitiser under the conditions of the test.