Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31.10.-18.11.2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was carried out in accordance with internationally valid GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Centralit- Substance type: pure substance- Physical state: white crystalline powder- Analytical purity: 99.9% (w/w)- Expiration date of the lot/batch: 16.9.2016- Stability under test conditions: stable- Storage condition of test material: in dry room at the temperature < 40°C

In vivo test system

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic- Age at study initiation: 8-10 weeks- Weight at study initiation: 16.6-19.22 g- Housing: maximum six animals in macrolon cages with sterilized softwood shavings- Diet (e.g. ad libitum): Pelleted standard diet for experimental animals ad libitum- Water (e.g. ad libitum): Drinking tap water ad libitum- Acclimation period: at least 5daysENVIRONMENTAL CONDITIONS- Temperature (°C): 22 +/- 3°C, permanently monitored- Humidity (%): 30 – 70 %, permanently monitored- Air changes (per hr): approximately 15 air changes per hours- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycleSTUDY TIME SCHEDULEAnimal arrival/ start of acclimatization: 12. 10. 2011Pilot experiment: 31.10.- 3.11. 2011Main study:First day of administration: 9.11.2011End of treatment period: 11.11.2011Application of radionuclide and necropsy: 14.11.2011

Study design: in vivo (LLNA)

Vehicle:
other: DAE 433 - mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol
Concentration:
The test substance was administered in the form of suspension in DAE 433.Concentration in formulation:30% (w/v) - 300 mg/mL3% (w/v) - 30 mg/mL0.3% (w/v) - 3 mg/mL
No. of animals per dose:
5 animals
Details on study design:
PILOT EXPERIMENTThe highest concentration 30% (maximum technically practicable concentration) was administered to three animals to assess and/or discard a possible systemic toxicity or high irritation of skin. During the pilot experiment no test item related effects were found in all three animals, respectively no clinical symptoms of systemic toxicity and no macroscopic changes (after necropsy) were detected. MAIN STUDYANIMAL ASSIGNMENT AND TREATMENTAnimals were subjected to a clinical examination (health check) shortly after arrival. No clinical changes were recorded.Study animals were randomly allocated to the dose groups manually and assigned animal numbers.TREATMENT PREPARATION AND ADMINISTRATION:Dosage volume: 25μL / ear / animalPreparation for administration: All suspensions were prepared by mixing an appropriate amount of test substance and the vehicle to obtain the application form in concentration of 30%, 3% or 0.3% (w/v). The suspensions were prepared before the start of application by mixing on magnetic stirrer and then were still mixed during application.Frequency of preparation: On each day immediately before administration.IN VIVO EXAMINATION- mortality- clinical observation- body weightPOST MORTEM INVESTIGATIONS- ears weights- incorporation of 3H-methyl thymidineDATA ANALYSISMean values and standard deviations of ears weight and incorporation of 3H-methyl thymidine were computed for the test item groups and for the positive as well as the vehicle control group. Stimulation index (for incorporation of 3H-methyl thymidine) was calculated by dividing mean values from the test substance groups and the positive control group by the corresponding mean value of the vehicle control group. The index for the vehicle control group was set at 1 by definition.
Positive control substance(s):
other: dinitrochlorobenzene (DNCB)
Statistics:
For statistical calculations the software Statgraphic ® Centurion (version XV, USA) was used. Statistical evaluation of measured parameters was performed at first by applying the non-parametric Kruskal-Wallis test for the comparison of the measured effect in all treatment groups with the vehicle control group, as global test, and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) for all two-group comparisons.

Results and discussion

Positive control results:
The positive control substance DNCB produced positive LLNA response at an exposure level expected to give an increase in the Stimulation Index SI ≥ 3 over the negative control group, which was in congruence with the expected mode of action of a contact allergen. The positive control also elicited a reaction pattern with statistically significant increase in ear weight. These results demonstrate that the method performed in conditions of laboratory has sufficient reliability.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI for the test groups treated by the test substance at all three dose levels below the threshold.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See Table 6

Any other information on results incl. tables

Table 6.  Summary of results

Group

Radioisotope incorporation

Ear weight

Mean DPM

SI

Mean (mg)

NC

252.80

1.00

22,98

PC

3393.15

13.42+

36,12*

30%

235.35

0.93

23,50

3%

356.31

1.41

23,50

0.3%

267.84

1.06

23,00

Figures with asterisk = values statistically significant on probability level < 0.05 (Mann-Whitney test)

Figures with cross = values ≥ 3

NC – Negative control group

PC – Positive control group

DISCUSSION

The animals exposed to the test substance showed no skin reactions and clinical symptoms of intoxication throughout the experiment.

The test substance did not show a tendency to increase ear weight – it means the test substance did not cause irritation of skin.

The comparison of the Stimulation Indexes between the treated groups and the control group revealed that the test substance did not cause a significant increase in radioisotope incorporation into the DNA of dividing lymphocytes.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated informationCriteria used for interpretation of results: EU
Conclusions:
Under the given test conditions, the test substance, Centralit, does not elicit sensitising response in LLNA assay.
Executive summary:

The test substance, Centralit, was tested for the assessment of skin sensitisation potential with the murine local lymph node assay. This study is a part of the test substance health hazard evaluation. The Local Lymph Node Assay (LLNA) with radionuclides was used. The testing was conducted according to the EU Method B.42, Skin sensitization: Local Lymph Node Assay, Council Regulation (EC) No. 440/2008, published in O.J. L142, 2008.

In this study the contact allergenic potential of Centralit was evaluated after topical application to female BALB/c mice. Five mice per group were exposed on the dorsum of both ears once a day by test substance and control substances during 3 consecutive days. Primary proliferation of lymphocytes in the lymph node draining the site of application was evaluated on the base on using of radioactive labelling. The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index, was determined. Statistical evaluation of ear weight was performed for elimination of false positive findings with certain skin irritants.

Concentrations: positive control DNCB (dinitrochlorobenzene): 0.5% (w/v) and Centralit: 30%, 3%, 0.3% (w/v) in DAE 433.

The animals exposed to the test substance at all concentrations showed no pathological skin reactions and no other negative clinical symptoms of intoxication throughout the experiment. There was no significant difference in body weight increment of all groups in comparison to the vehicle control.

The positive control substance DNCB elicited a reaction pattern with statistically significant increase in ear weight and Stimulation Index of cell proliferation 13.42, which was in congruence with his expected mode of action as a contact allergen.

The test substance did not show a tendency to increased ear weight in any of concentrations tested. The result of skin irritation effect was considered as negative – it means the test substance did not cause irritation of skin.

Comparison of Stimulation Indexes between all treated groups and control vehicle group revealed that the test substance Centralit did not cause significant increase in radioisotope incorporation into the DNA of dividing lymphocytes.

In conclusion, at the given experimental conditions the test substance Centralit provided a negative result in LLNA test.