Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 April to 31 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary GLP guideline-compliant study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (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
Details on test material:
Calcium fluoride, a white powder, test substance no. 10/0041-1, batch no. F1510740 835, purity > 98%. The test substance was homogeniuos by visual inspection, and stability under storage conditions over the study period was guaranteed by the sponsor.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
The animals were female CBA/J mice obtained from Charles River Laboratories (Germany), aged 8-12 weeks and weighing 17.7-21.1 g on study day 0. They were acclimatised for 14 days. Mice were singly housed in Makrolon type II cages with Lignocel PS14 bedding (SSNIFF), and identified by cage cards. The animal rooms were fully air-conditioned, with a temperature range of 20-24°C and relative humidity of 30-70%. Light was provided on a 12 hour light/dark cycle. Each mouse was provided with a PLEXX mouse tunnel and Nestlets NES 3600 as enrichment. They were fed Kliba-Labordiat (Maus/Ratte Haltung "GLP"; Provimi Kliba, Switzerland) ad libitum. Tap water was also provided ad libitum.
Food water and bedding were analysed for known contaminants and all found to be within acceptable limits.

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
40% solution in propylene glycol.
No. of animals per dose:
5
Details on study design:
A solubility experiment was performed, and it was found that the highest test-substance concentrations which could be technically used was a 40% test substance preparation in propylene glycol. Good solubility of the preparation was achieved at this concentration. The stability of the test substance in the vehicle was determined by the concentration control analysis. Samples taken were stored at room temperature over the maximum duration of the application period and were subsequently deep-frozen. The concentration of the test substance in the vehicle was determined once during the study.
The highest test-substance concentration not inducing local signs of irritation and/or systemic toxicity was determined in a pre-test using 3 mice. The mice were treated with the 40% preparation on 3 consecutive days. The ears were punched afeter sacrifice at the apical area using a biopsy punch and were immediately pooled per animal and weighed. The weight of the pooled lymph nodes from both sides were determined for each animal. There was no relevant increase in lymph node weights, but some increase in ear weights, indicative of slight irritation. There were no signs of systemic toxicity. As 40% was the maximum achievable technical concentration, it was chosen for the main study, which was carried out as a limit test.

Mice were randomised to control and test groups according to the algorithms of Nijenhuis and Wilf (1978).
Individual body weights were recorded on day 0 prior to the first application and on day 5 prior to sacrifice. Mice were observed for mortality twice each workday, and one at weekends and on public holidays.
The substance was applied epicutaneously; 25 µl per ear on the dorsal surface of both ears. The test substance was applied on 3 consecutive days to the same application site (day 0-2). Controls received the vehicle only.
On study day 5, (approx. 66-72 h after the last test substane application), the mice were injetced i.v. with 20 µCi of ³H-thymidine in 250 µl of sterile saline into a tail vein. The mice were sacrificed on study day 5 about 5 hours after ³H-thymidine injection by cervical dislocation under isoflurane anaesthesia. Immediately after the death of each animal a circular piece of tissue (diameter 0.8 cm) was ounched out of the apical part of the ear of all animals. The weight of the pooled punches was determined for each test group. The measures serve to detect potential inflammatory ear swelling. Immediately after removal of the ear punches the left and right auricular lymph nodes from both sides were dissected. The weight of the pooled lymph nodes from both sides was determined for each mouse.
After weight determination, the pooled lymph nodes of each group were stored in PBS in an ice-water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing all lymph nodes per test group through an iron mesh (200 µm) into 40 ml of PBS. An aliquot of each suspension was further diluted with Casy-ton in a ratio of 1:500 and cell count determined using a Casy-Counter.
The remaining cell suspensions were washed twice with PBS and precipitated with 5% trichloro-acetic acid. Each precipitate was transferred to scintillation fluid and incorporation of ³H-thymidine was measured in a ß-scintillation counter.

The stimulation indices of cell count, ³H-thymidine incorporation, lymph node weight and ear weight were calculated as the ratio of the test group values for these parameters divided by those of the control vehicle group.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Formal statistical analysis not required.

Results and discussion

Positive control results:
Alpha-hexylcinnamise produced consistently positive results in the test laboratory with CBA/J mice.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Stimulation indices were calculated using historical vehicle control data: SI (control) = 1.00, SI (40% formulation) = 1.10.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Historical control mean = 561.2 DPM. Concurrent control mean = 1380.9 DPM. Test group 40% formulation mean = 2299.7 DPM.

Any other information on results incl. tables

The stability of the test substance in the vehicle was confirmed indirectly by analysis of the concentration. The test substance preparations were visually homogenous. The concentration analysis revealed an analytical value of approximately 120% of the nominal concentration.

The pre-test with the 40% formulation caused no relevant increase in lymph node weights, but some increase in ear weights indicative of ear irritation. No signs of toxicity were observed.

The 40% test substance preparation did not induce increases in ³H-thymidine incorporation or in auricular lymph node cell counts. There was no increase in lymph node weights. The lymph node parameters of the control group were considerably higher than the mean historical control data for the vehicle propylene glycol, therefore all stimulation indices were also calculated in relation to mean historical control values. The stimulation indices calculated in relation to the historical control values show a biologically relevant increase of ³H-thymidine incorporation, however this did not match the less pronounced increases in cell count and lymph node weight. Thus the increase in ³H-thymidine incorporation was attributed to the joint occurrence of a high concurrent control value as compared to the historical control.

Mean body weights of both test groups were slight reduced over the study period. No abnormalities were observed during general observation.

Data for the main test, and historical control data are presented in the following table.

Treatment

Cell counts

(Counts/Lymph Node Pair)

Stimulation Index

Propylene glycol vehicle historical control (mean)

8634667

6809867

1.00a

40% test substance in vehicle

8573333

0.99

1.26

 

 

 

Treatment

³H-thymidine incorporation

(DPM/Lymph Node Pair)

Stimulation Index

Propylene glycol vehicle historical control (mean)

1380.9

561.2

1.00a

40% test substance in vehicle

2299.7

1.67a

4.10b

 

 

 

Treatment

Lymph Node Weight

(mg/Lymph Node Pair)

Stimulation Index

Propylene glycol vehicle historical control (mean)

5.4

4.5

1.00a

40% test substance in vehicle

5.4

1.01a

1.20b

 

 

 

Treatment

Ear Weight

(mg/animal)

Stimulation Index

Propylene glycol vehicle historical control (mean)

32.6

31.9

1.00a

40% test substance in vehicle

32.7

1.00a

1.02b

 

a  test group / historical vehicle control

b  Stimulation index (SI) calculated using the historical control data

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
It was concluded that calcium fluoride is not sensitising in the mouse LLNA.
Executive summary:

The skin sensitising potential of calcium fluoride was determined in the Murine Local Lymph Node Assay, according to OECD 429.

Groups of 5 female CBA/J mice were treated on each ear with 25 µl of a 40% w/w preparation of the test substance in propylene glycol, or the vehicle alone, for 3 consecutive days. 40% was the maximum achievable concentration, therefore the study was conducted as a limit test.

Three days after the last application the mice were injected i.v. with ³H-thymidine, and were sacrificed approximately 5 hours after injection for collection of the auricular lymph nodes. The weights of the lymph nodes were determined per animal, then pooled per group for determination of cellular content and ³H-thymidine incorporation. An area with a diameter of 0.8 cm was removed from each ear and pooledd weights per test group were determined (as an indicator of skin irritation).

Compared to the concurrent vehicle control values, the test substance (40% preparation in propylene glycol) did not cause increases in ³H-thymidine incorporation or in the auricular lymph node cell counts. There was no increase in lymph node weights or ear weights.

The lymph node parameters of the concurrent control group were considerably higher than the mean historical control data for this vehicle, therefore all stimulation indices were additionally calculated in relation to the mean historical values.

The SI values calculated using the historical control values showed a biologically relevant increase of ³H-thymidine incorporation, but this did not match the less pronunced increases in cell count and lymph node weight. Therefore the increase in ³H-thymidine incorporation was attributed to the occurrence of a high concurrent control value as compard to the historical control.

It was concluded that calcium fluoride is not sensitising in the LLNA, under the conditions of the study.