Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 to 30 June 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant with OECD 429 and GLP guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

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:
Name: Thiazol Blau

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Species and strain: CBA/J@Rj mice
- Source: ELEVAGE JANVIER Route des Chènes Secs B.P. 4105, 53940 LE GENEST-ST-ISLE, France.
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: 21.3 – 22.9 grams
- Housing: single
- Diet: ssniff SM R/M-Z+H ad libitum
- Water: tap water ad libitum
- Acclimation period: 27 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 24 to 30 June 2009

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
1, 2.5, 5%
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: max 5% in DMSO
- Irritation: no
- Lymph node proliferation response: not determined


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Lymph node proliferation assay with incorporated tritiated thymidine
- Criteria used to consider a positive response:
-- exposure to at least one concentration resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
AND
-- 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:
Each mouse was topically dosed with 25 µl of the appropriate formulation (1%, 2.5%, 5%) of the test item using a pipette, on the dorsal surface of each ear.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Compared to pooled control values of 3 parallel running tests: 1.0%: 2.0 2.5%: 2.9 5.0%: 2.1
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 1.0%: 2828.5 2.5%: 4214.5 5.0%: 2903.5

Any other information on results incl. tables

The test item was agreen blue, nearly black powder, which was dissolved in the selected vehicle (DMSO). Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay.According to evaluation criteria of the OECD Guideline 429 the proliferation value above 3 at concentration of 2.5% lies above the threshold value for a positive response, however the results do not meet the criteria of being “compatible with a conventional dose response” (see A in table below). To clarify the sensitisation capacity of the test item, the measured proliferation values have been compared to the average proliferation value of the three parallel negative control values of studies 09/069-037E (DPN = 218.7), 09/068-0378E (DPN = 150.3) and 09/067-037E (DPN = 158.4) (see B in table below). It is considered valid to take the 3 control groups into account since they were all on the same batch of animals, run in parallel, with the same control solvent. Furthermore, the control values in these studies were towards the low end of the historic control data for this solvent (see Appendix 5), confirming the control result in this case was lower than typically expected.

Comparison of Stimulation Index Values for different negative control values

Test Group Name

DPN

Stimulation Index Values A

Stimulation Index Values B

A

Negative control DMSO

150.3

-

-

B

Mean of negative controls DMSO

175.8

-

-

Thiazol Blau 5 (w/v) %

353.6

2.4

2.0

Thiazol Blau 2.5 (w/v) %

526.8

3.5

 2.99

Thiazol Blau 1 (w/v) %

362.9

2.4

2.1

Positive control 25 (w/v) % HCA

1052.8

7.0

6.0

  

This table gives further evidence that Thiazol Blau should not be classified as a sensitiser, as the stimulation indices for the mid dose calculated as the mean of the parallel running negative controls for DMSO lies below the threshold value of 3, whereas the positive control was unequivocally identified as a sensitizer.

The lack of a biological dose response and the fact that the highest value falls below a realistic threshold of 3 times the mean control value for the parallel studies, indicates that there was no positive effect from treatment with Thiazol Blau.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In conclusion, under the conditions of the present assay, Thiazol Blau, tested in a suitable vehicle, proved to have no sensitizing potential (non-sensitizer) in the Local Lymph Node Assay.
Executive summary:

The aim of this study was to determine the skin sensitisation potential of Thiazol Blau following dermal exposure.

Based on results of the Preliminary Compatibility Test and on recommendations of the OECD Test Guideline 429, the test item was dissolved in Dimethyl-sulphoxide (DMSO). The maximum attainable concentration based on solubility of the test item in DMSO was 5%.

A Preliminary Irritation/Toxicity Test was performed with the test item at concentrations of 5% and 2.5% in the selected vehicle. According to this test, the applicability and biocompatibility of the test item to the animals’ ears (2 female CBA/J@Rj mice/dose) at the maximum concentration of 5 % was acceptable.

 

In the main assay twenty female CBA/J@Rj mice were allocated to five groups of four animals each:

- three groups received the appropriate formulation of Thiazol Blau at concentrations of 5%, 2.5% and 1%,

- the negative control group received the vehicle (DMSO),

- the positive control group received 25% alpha-Hexylcinnamaldehyde (HCA) in DMSO.

 

The test item solutions were applied to the dorsal surface of both ears of the mice (25 µL/ear) for three consecutive days (Days 1, 2, and 3). There was no treatment on Days 4, 5, and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

 

No mortality or systemic clinical signs were observed during the study. No treatment related effects were observed on animal body weights in any of the treated groups. No cutaneous reactions were observed at the site of the treatment in any of the treated groups.

 

The stimulation index (SI) values of the test item were 2.4, 3.5, and 2.4 at treatment concentrations of 5%, 2.5%, and 1%, respectively. The response went above the threshold factor of 3, but the results did not show a clear conventional dose response.

This apparently positive response (SI >= 3) for the intermediate dose level is due to the very low DNP values (150.3) of the vehicle control which lies at the lower end (133.3) of the historical control range. Calculating the SI values for Thiazol Blau with the mean control DPN of the three parallel studies, leads to SI values below 3 for all concentrations tested.

Consequently, Thiazol Blau does not meet the criteria for a sensitiser.

 

A significant lymphoproliferative response (stimulation index value of 7.0) was noted for the positive control chemical HCA. This holds also true for calculating the SI value with the mean DPN value (6.0). This result confirmed the validity of the assay.

 

In conclusion, under the conditions of the present assay, Thiazol Blau, tested in a suitable vehicle, proved to have no sensitizing potential (non-sensitizer) in the Local Lymph Node Assay.