Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 23,2012 to August 7,2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant with international guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
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
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

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
- Starain: CBYJICO
- Age at study initiation:8 to 10 weeks
- Weight at study initiation:17.53 – 19.68 g
- Housing:maximum six in macrolon cages (35x20x15 cm) with sterilized softwood shavings
- Diet: Pelleted standard diet for experimental animals ad libitum (ST 1 BERGMAN) Microbiological control and content of nutrients is performed acco rding internal SOP No. 72.
- Water : Drinking tap water ad libitum
- Acclimation period:5 days
- Health examination: All animals were examined during the acclimatisation period
- Prophylactic arrangement: Cleaning and disinfection of animal room was regularly performed, as it is described in internal SOP No.10.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 + 3°C, permanently monitored
- Humidity: 45.7 – 60.8 %, permanently monitored
- Photoperiod : 12 hours light/dark cycle: 6am-6pm/6pm-6am

Study design: in vivo (LLNA)

Vehicle:
other: DAE 433
Concentration:
vehicle: DAE 433 - mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol
Application form of the test substance
The test substance was administered in the form of suspension in DAE 433.
Concentration in formulation:
30% (w/v) 300 mg/mL
3% (w/v) 30 mg/mL
0.3% (w/v) 3 mg/mL

All suspensions were prepared by mixing an appropriate amount of DIRECT BROWN 44 (1.5g, 0,15g or 0.015g) and the vehicle to obtain the application form (3x5mL) 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 stili mixed during application.
Frequency of preparation: On each day immediately before administration (10-20 min)

Dosage volume: 25μL / ear / animal
No. of animals per dose:
Pilot experiment: 3 females
Exposed groups: 15 females (5 animals in three groups)
Positive control group: 5 females
Negative control group: 5 females
Reserve group – 2 females (Reserve animals were intended for possible replacement of the animals in other groups during the acclimatisation period)
Total: 30 animals


Group 1 Group 2 Group 3 Group 4 Group 5 Group 6
Vehicle Positive Test Test Test Reserve animals
control substance substance substance
stance
Concentration % 0 0.5 30 3 0.3 -
Number of animals 5 5 5 5 5 2
Animal No. 1-5 6-10 11-15 16-20 21-25 26-27
Cage No. 1 2 3 4 5 6
Details on study design:
RANGE FINDING TESTS:
- Irritation: The highest concentration 30% was administered to three animals to assess and/or discard a possible systemic toxicity or high irritation of skin.


MAIN STUDY
- Clinical observations: Clinical signs were assessed using a defined scoring system described in SOP M/8 – twice daily during the dosing period. Efforts were made to characterize onset and duration of signs observed.
- Body weight:
Individual body weights were measured using an electronic balance. Weighing was performed on the first day of treatment, before dosing, and on the day of necropsy before application of the radionuclide.


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

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The SI for the test groups treated by the test substance at all three dose levels are below the threshold, stimulation index (SI) is < 3 . So the result of LLNA assay is negative. The test substance showed a tendency to form residues. Residues of the test substance on the ears caused this weight increase. In other groups of animals administered with test substance, a statistically significantly weight increase of ear targets was not found out – it means the test substance did not cause irritation of skin
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM Negative control:328.10 Positive control:3266.69 Test item at 30%: 368.57 Test item at 3%:250.67 Test item at 0.3%: 176.67

Any other information on results incl. tables

Necropsy

The third day after last administration (five hours after application of radionuclide), all test animal were sacrificed by injection of veterinary preparation T 61 (0,5 mL i.p.)

Test validity criteria

The test is considered valid, if the positive control substance DNCB will produce positive LLNA response and if the stimulation index SI is ≥ 3 over the negative control group.

Clinical observation:

No animal died during the main experiment. No symptoms of toxicity and no erythema on application site were observed in animals from the negative control group and in groups administered by the test substance. All animals in the positive control group showed symptoms caused by the application of DNCB: hyperaemia of skin with well defined erythema on application site, clonospasm and decreased response to stimuli.

Body weight:

Individual body weight of females before administration and before necropsy was relatively well balanced. ln the positive control group, the weight of ear target was statistically significantly increased against negative control group. Statistically significantly increase ear weight was recorded only in animals at the highest dose level (30% test substance) but without lymphocytes proliferation and skin irritation. The test substance DIRECT BROWN 44 showed a tendency to fortn residues. Residues of the test substance on the ears might have caused this weight increase. In other groups of animals administered with test substance, a statistically significantly weight increase of ear targets was not found out.

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 (=0.5 cm^2) were excised using a disposable punch and weighed together on analytical scales

Incorporation of 3H-methyl thymidine

The tissue of both of the lymph nodes was prepared by gentle mechanical disaggregation through 100 µm-mesh nylon gauze with concomitant pooling in 1 mL PSB (Phosphate Buffered Saline). The pairs of lymph nodes was analysed separately for each animal per group. Cells were washed twice with an excess of PBS and precipitated with 5% trichloroacetic acid (TCA) at 4 °C for 18h. The pellets were re-suspended in 1 mL TCA and transferred to scintillation vials containing 10 mL of scintillation fluid. Incorporation of 3H-methyl thymidine were measured by (3-scintillation counting (Beckmann LS 6500) as disintegrations per minute (DPM).

validity criteria

The test is considered valid, if the positive control substance DNCB will produce positive LLNA response and if the stimulation index SI is ≥ 3 oves the negative control group.

SUMMARY TABLE

Group Radioisotope incorporation Ear weight
Mean DPM SI Mean (mg)
NC 328.1 1.00 23.38
PC 3266.69 9.96+ 38.24*
30% 368.57 1.12 24.82*
3% 250.67 0.76 23.66
0.30% 176.67 0.54 23.44

The comparison of the Stimulation h dexcs between the treated groups and the control group revealed that the test substance DIRECT BROWN 44 did not cause a significant inerease in radioisotope incorporation iato the DNA of dividing lyinphocytes.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the given test conditions, the test substance, DIRECT BROWN 44, does not elicit sensitising response in LLNA assay.
Executive summary:

The test substance, Direct Brown 44, 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 Method B.42, Skin sensitisation: Local Lymph Node Assay, Council Regulation (EC) No.440/2008, Published in O.J. L142, 2008.

In this study the contact allergenic potential of DIRECT BROWN 44 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 DIRECT BROWN 44: 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 9.96, which was in congruence with his expected mode of action as a contact allergen.

The test substance DIRECT BROWN 44 showed a tendency to increase of ear weight in the highest dose level but without lymphocyte proliferation and skin irritation. Residues of the test substance on the ears caused this weight increase. 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 DIRECT BROWN 44 did not cause significant increase in radioisotope incorporation into the DNA of dividing lymphocytes.

In conclusion, at the given experimental conditions the test substance DIRECT BROWN 44 provided a negative result in LLNA test.