Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
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): Tar, brown-coal, low-temp.
- 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: viscous liquid
- Lot/batch No.: T201E
- Expiration date of the lot/batch: 31.3.2011
- Stability under test conditions: stable
- Storage condition of test material: The substance was stored in closed vessel in dry room at the temperature bellow 25ºC in dark.

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: 18.3 - 21.9 g
- Housing: group-wise five 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% (v/v)
3% (v/v)
0.3% (v/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:
PILOT TEST:
The highest concentration 30% was administered to three animals to assess of systemic toxicity. 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 STUDY
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 allowes to access 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.

Evaluation of results
Stimulation index (SI)
The SI is obtained by dividing the pooled radioactive incorporation for each treatment group by the incorporation of the pooled vehicle control group; this yields a mean SI.
The response towards the test substance is considered positive, if the stimulation index (SI) is ≥ 3, and the response increases in dose-related manner (dose-response relationship).
The response is considered negative, if the stimulation index (SI) is < 3 without the dose–response relationship.
The response is considered ambiguous if the stimulation index (SI) is < 3, but the response increases in dose-related manner (dose–response relationship), and eventually statistical significance is observed.
Validity criteria:
The test is considered valid, if the positive control substance DNCB (dinitrochlorobenzene) produces a positive LLNA response, and if the stimulation index SI is ≥ 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.
A 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.

TREATMENT PREPARATION AND ADMINISTRATION
All suspensions were prepared by mixing an appropriate amount of the substance and the vehicle to obtain the application form in concentration of 30%, 3% or 0.3% (v/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. The sixth day injection of 250 μL of phosphate-buffered saline (PBS) containing 7.18 x10e5 Bq of 3H-methyl thymidine into all test and control mice via the tail vein.
Five hours later, the animals were killed.

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 ear weights and incorporation of 3H-methyl thymidine into the adjacent lymph nodes were computed for the test substance treatment groups and for the positive as well as the vehicle control group. Results (incorporation of 3H-methyl thymidine) for each treatment group were expressed as mean Stimulation Index (SI). The SI was the ratio of the mean dpm/mouse within each test substance treatment group versus the mean dpm/mouse for the vehicle treated 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. At first the global comparison of all three values of the concentration groups with vehicle control is performed by applying the non-parametric Kruskal-Wallis test, and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) was applied to all two-group comparisons.

Results and discussion

Positive control results:
In the positive control group, the SI was ≥ 3 (11.55) – test LLNA was efficient.

Irritating effect:
In the positive control group, the weight of ear target was statistically significantly increased as compared to the negative control group – the test design used is efficient in the detection of irritation effect.

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

Table: Radioisotope incorporation and ear weight:

Group

        Radioisotope incorporation

Ear weight

 

Mean DPM

SI

Mean (mg)

NC

601.28

1.00

23.18

PC

6945.51

11.55+

32.26*

30%

7466.35

12.42+

37.14*

3%

2443.74

4.06+

24.54*

0.3%

854.77

1.42

23.26

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

DPM - Disintegrations Per Minute

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
The positive control substance DNCB produced a positive LLNA response at an exposure level expected to give an increase in the Stimulation Index SI of ≥ 3 over the negative control group, which was consistent with the expected mode of action of a contact allergen. The positive control also elicited a reaction pattern with a statistically significant increase in ear weight. These results demonstrate that the method had sufficient sensitivity.

The animals exposed to the test substance at all dose levels showed no pathological skin reactions.
No symptoms of systemic toxicity were observed at all dose levels.
At the highest and middle dose levels, statistically significant increases in the weight of ears were recorded. The result of skin irritation effect was considered as positive – it means the test substance caused irritation of skin.

The comparison of the Stimulation Indexes between the treated groups and the control group revealed that the test substance caused a significant increase in radioisotope incorporation into the DNA of dividing lymphocytes.

Executive summary:

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

Concentrations:

positive control DNCB (dinitrochlorobenzene): 0.5% (w/v) and

test substance: 30%, 3%, 0.3% (v/v) in the solvent mixture, DAE 433.

 

The animals exposed to the test substance at all dose levels showed no pathological skin reactions. No symptoms of systemic toxicity were observed at all dose levels. The positive control substance DNCB elicited a reaction pattern with statistically significant increase in ear weight and cell proliferation, the Stimulation Index reaching 11.55, which was consistent with its expected mode of action as a contact allergen.

 

The test substance showed a tendency to increased ear weight in the highest and middle of concentrations tested. The result of skin irritation effect was considered as positive – it means the test substance caused irritation of skin.         

The comparison of Stimulation Indexes between the treated groups and the control group revealed that the test substance caused a significant increase in radioisotope incorporation into the DNA of dividing lymphocytes. This effect was dose dependent.

 

Based on the positive results of LLNA study, the test substance may be a potential contact allergen, but the irritation effect observed does not rule out the possibility that it may be a false positive result.