Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 27, 2010 to August 11, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Performed under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): crystalline calcium silicate hydrate (xonotlite - tobermorite)
- Physical state: Solid
- Analytical purity: Confidential
- Lot/batch No.: Confidential
- Storage condition of test material: At room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst, The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 19.5-23.8 g
- Housing: Individually, under standard laboratory conditions
- Diet: Ad libitum, pelleted standard diet
- Water: Ad libitum, tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 45-94
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
other: Ethanol:sterile water (3+7)
Concentration:
2.5, 5, and 10% (highest concentration which was technically possible )
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which could be technically used was a 10 % suspension in ethanol:sterile water (3+7). In all other vehicles (acetone:olive oil (4+1), dimethylformamide, methyl ethyl ketone, propylene glycol, dimethylsulfoxide, and ethanol:sterile water (7+3)) tested the test item could not be formulated.
- Irritation: Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with concentrations of 5 and 10% each on 3 consecutive days. In the pre-test clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 7. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response:
1. Exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index (SI), and
2. 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: For the highest concentration 100 mg of the test item were placed into an appropriate container on a tared balance and 630 mg sterile water were added. The suspension was boiled up for a short time. After cooling, 270 mg of ethanol were added. The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion. Formulation was applied to the dorsal ears on 3 consecutive days, performed according to guideline (proliferative capacity was determined by incorporation of 20.3 uCi 3H-methyl thymidine per mouse).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean values and standard deviation were calculated for the body weights.

Results and discussion

Positive control results:
Calculated S.I. values for 0%, 5%, 10% and 25% hexyl cinnamic aldehyde were 1.00, 2.04, 3.41 and 6.14 resp. An EC3 value of 8.5% (w/v) was calculated. This proves the reliability of the model, as the EC3 value is within the acceptable range.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.4
Test group / Remarks:
2.5%
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
1.23
Test group / Remarks:
10%
Cellular proliferation data / Observations:
Disintegrations per minute (DPM):
Control: 1778 dpm
2.5 %: 2486 dpm
5 %: 1961 dpm
10 %: 2179 dpm

Any other information on results incl. tables

- No deaths were noted during study period.

- No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

- Body weight was within the range commonly recorded for animals of this strain and age.

- An EC3 value could not be calculated for Crystalline calcium silicate hydrates (xonotlite - tobermorite), since all obtained SI´s were below 3.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
In this Local Lymph Node Assay, Stimulation Indices (SI) of 1.40, 1.10, and 1.23 were determined when the test item was applied to the skin at concentrations of 2.5, 5, and 10% in ethanol:sterile water (3+7). Under the conditions of this study crystalline calcium silicate hydrates (xonotlite - tobermorite) did not induce sensitisation in the mouse. The highest SI calculated was 1.40, which is below the threshold of 3 for classification. Therefore, the test substance does not need to be classified for sensitisation based on the criteria outlined in Annex I of 1272/2008/EC .
Executive summary:

This Local Lymph Node Assay (OECD 429) was performed to determine the sensitising potential of crystalline calcium silicate hydrates (xonotlite – tobermorite) in mice. Test concentrations of 2.5%, 5%, and 10% in ethanol:sterile water (3+7) were used (highest technically achievable concentration). Alpha-hexylcinnamicaldehyde was used as positive control substance. Proliferative capacity was determined by incorporation of 20.3 uCi 3H-methyl thymidine per mouse and subsequent liquid scintillation counting. Mortality, clinical signs and body weight were recorded.

No mortality or clinical signs were noted and body weights were within the range commonly recorded for the test animals. The amount of disintegrations per minute (DPM) that were measured by liquid scintillation counting were (after correction for background counts): 1778, 2486, 1961, and 2179 for the concentrations of 0%, 2.5%, 5, and 10%, resp. Accompanying Stimulation Indices (SI) that were calculated: 1.40, 1.10, and 1.23 for 2.5%, 5%, and 10%, resp. An EC3 value could not be calculated for the test item, since all obtained SI´s were below 3.

Under the conditions of this study crystalline calcium silicate hydrates (xonotlite – tobermorite) did not induce sensitisation in the mouse. The highest SI calculated was 1.40, which is below the threshold of 3 for classification. Therefore, the test substance does not need to be classified for sensitisation based on the criteria outlined in Annex I of 1272/2008/EC .