Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31.3.2010 - 14.5.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out in accordance with internationally valid GLP principles.

Data source

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

Materials and methods

Test guideline
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
Details on test material:
- Name of test material (as cited in study report): Reaction product of Distillates (petroleum), acid-treated heavy naphthenic and calcium oxide
- Molecular formula: not known - UVCB substance
- Molecular weight: not known - UVCB substance
- Smiles notation: not known - UVCB substance
- InChl: not known - UVCB substance
- Substance type: technical product
- Physical state: solid
- Lot/batch No.: 26.2.2010
- Expiration date of the lot/batch: 26.2.2012
- Stability under test conditions: stable
- Storage condition of test material: The substance was stored in PE bottle at room temperature.

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, RČH CZ 21760118
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: 19 - 21.7 g
- Housing: Animals in groups of maximum six in macrolon cages with sterilized softwood shavings
- Diet: Pelleted standard diet for experimental animals ad libitum
- Water: Drinking tap water ad libitum.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: Room temperature 22 +/- 3ºC, permanently monitored
- Humidity: Relative humidity 30 - 70 %, permanently monitored
- Photoperiod: 12 hours light/dark cycle


Study design: in vivo (LLNA)

Vehicle:
other: DAE 433 - mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol
Concentration:
30% (w/v)
3% (w/v)
0.3% (w/v)

No. of animals per dose:
Pilot experiment: 3 females
Exposed groups: 15 females (5 animals in 3 groups)
Positive control group: 5 females
Negative control group: 5 females

Details on study design:
PRINCIPLE OF THE METHOD
The principle of the Local Lymph Node Assay (LLNA) is that sensitizers induce a primary proliferation of lymphocytes in the lymph node draining the site of chemicals application. The proliferation is proportional to the dose applied (and to the potency of the allergen) and provides a simple means of obtaining an objective, quantitative measurement of sensitisation. The LLNA assesses this proliferation as a dose-response relationship in which the proliferation in test groups is compared to that in vehicle treated controls, termed the Stimulation Index. The method is based on the use of radioactive labelling (3H-methyl thymidine) to measure cell proliferation.
- Criteria used to consider a positive response:
Cell proliferation: The response towards the test substance is considered positive, if the stimulation index (SI) ≥ 3, and the response increases in dose-related manner (dose-response relationship).
Validity criteria : The test is considered valid, if the positive control substance produces positive LLNA response and if the stimulation index SI ≥ 3 over the negative control group.
Ear weight – irritation effect: If after treatment with the test substance a statistically significant increase of ear weight together with clear concentration dependence of the effect is recorded, the inflammatory effect is considered as irritation induced by the test substance. Positive result in cell proliferation reveals that the test substance could be a contact allergen, but an irritation effect of test substance (increased ear weight) does not rule out the possibility that it can be a false positive result.

PILOT TEST
The 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. The route of administration was the same as in the main study. During the pilot experiment no test substance related effects were found in all three animals, respectively no clinical symptoms of systemic toxicity and no macroscopic changes (after necropsy) were detected.

MAIN TEST
TREATMENT PREPARATION AND ADMINISTRATION
All suspensions were prepared by mixing an appropriate amount of test item (1.5 g, 0.15 g or 0.015 g) 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.
The volume of the application form was constant at all groups of animals - 25 µL of the appropriate dilution to the dorsum of each ear once a day morning for 3 consecutive days. The application was performed very slowly by micropipette.

IN VIVO EXAMINATION
Health and mortality: at least twice daily during the dosing period
Clinical observations: at least twice daily during the dosing period
Body weight: on the first day of treatment, before dosing, and on the day of necropsy before application of the radionuclide

POST MORTEM INVESTIGATIONS
Ears weights: Immediately after death, the both ears were cut off and circular pieces from the apical area of each ear with a diameter of 8 mm were excised and weighed together.
Incorporation of 3H-methyl thymidine: were measured by β-scintillation counting as disintegrations per minute (DPM).

DATA ANALYSIS
Mean values and standard deviations of ears weight and incorporation of 3H-methyl thymidine were computed for the test substance 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: Dinitrochlorbenzene, 0.5% (w/v) solution. The vehicle and dosage volume were the same as in treated groups.
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:
Cell proliferation: In the positive control group, the SI was ≥ 3 (16.71) – the LLNA was efficient.
Irritating effect: In the positive control group, the weight of ear target was statistically significantly increased against negative control group.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see table bellow
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see table bellow

Any other information on results incl. tables

Test results (DPM, SI and ear weight)

 

Group

Radioisotope incorporation

Ear weight

mean (mg)

mean DPM

SI

NC

512.94

1.00

22.94

PC

8751.15*

16.71+

40.58*

30%

1122.22

2.19

24.58

3%

404.77

0.79

22.88

0.3%

618.40

1.21

21.86*

 

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

 

 

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test substance provided a negative result in LLNA test.

Executive summary:

The test substance was tested for the assessment of skin sensitisation potential with the murine local lymph node assay. 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 test item 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 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 (SI), was determined. Statistical evaluation of ear weight was performed for elimination of false positive findings with certain skin irritants.

Comparison of Stimulation Indexes between all treated groups (30%, 3%, 0.3% (w/v) in vehicle DAE 433) and control vehicle group revealed that the test substance did not cause increase in radioisotope incorporation into the DNA of dividing lymphocytes. 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.

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 16.71, which was in congruence with his expected mode of action as a contact allergen. Concentrations for positive control DNCB (dinitrochlorobenzene) were 0.5% (w/v).

At the given experimental conditions the test substance provided a negative result in LLNA test.