Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3.3.-22.3.2010
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
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Ashes (residues) – Biomass Combustion
- Physical state: solid
- Substance type: technical product
- Appearance: black powder

- Composition of test material, percentage of components:
SiO2 37.81 % (w/w), K2O 17.01 % (w/w), CaO (total) 11.92 % (w/w), P2O5 6.15 % (w/w), Al2O3 4.05 % (w/w), SO3 (sulphate) 3.47 % (w/w), MgO 2.25 % (w/w), Fe2O3 1.81 % (w/w), CaO (free) 1.40 % (w/w), CO2 1.29 % (w/w), Na2O 0.60 % (w/w), TiO2 0.34 % (w/w).
- Impurities (identity and concentrations):
Metals: As, Be, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Sb, Se, Tl, V, Zn sum <
0.1%

- Lot/batch No.: BMA/F/2009
- Expiration date of the lot/batch: 15 years / 12/2024
- Storage condition of test material: PE container, temperature bellow 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, RČH CZ 21760118
- Age at study initiation: 8-10 weeks (at start of dosing)
- Weight at study initiation: 19.68 to 21.82 g (at start of dosing)
- Housing: group-wise five in macrolon cages with sterilized softwood shavings.
- Diet (e.g. ad libitum): Pelleted standard diet for experimental animals ad libitum. Microbiological control and content of nutrients was performed.
- Water (e.g. ad libitum): Drinking tap water ad libitum.
- Acclimation period: at least 5 days
- Prophylactic arrangement: Cleaning and disinfection of animal room was regularly performed, as it is described in corresponding SOP No.10.
- Cage identification: By cage number, study number and group specific colour.
- Animal identification: By felt tip marking (from 1 to 5 per each group and group specific colour).
- Random selection: According to SOP No.42.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C, permanently monitored
- Humidity (%): 30 – 70 %, permanently monitored
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

STUDY TIME SCHEDULE
Animal arrival/ start of acclimatization: 3.3.2010
Pilot experiment: 9.-12.3.2010
First day of administration: 17.3.2010
End of treatment period: 19.3.2010
Necropsy: 22.3.2010

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/mL
3% (w/v) 30 mg/mL
0.3% (w/v) 3 mg/mL
No. of animals per dose:
Exposed groups - 15 females (5 animals per concentration)
Details on study design:
PILOT EXPERIMENT
The highest concentration 30% was administered to three animals to assess a possible systemic toxicity. The route of administration was same as in the main study. During pilot experiment no clinical symptoms of systemic toxicity and no macroscopic changes (after necropsy) were found out in all three animals.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Animals were subjected to a clinical examination (health check) shortly after arrival.
Study animals were randomly allocated to the dose groups manually and assigned animal numbers.

- Criteria used to consider a positive response:
The results of the LLNA were evaluated according to the following criteria.

Stimulation index
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 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 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:
- Dosage volume: 25μL / ear / animal
- Preparation for administration: All solutions were prepared by dissolving an appropriate amount of Ashes (residues) in the vehicle to obtain a concentration of 30%, 3% or 0.3% (w/v). Before start of application the suspension was mixed for 5 minutes with a magnetic stirrer.
- Frequency of preparation: each day of administration.
- Application: The volume of the application form was constant at all groups of animals - 25μL 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 to avoid losses caused by draining from the ear.
- 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:
Immediately after death, both ears of one animal were cut off and circular pieces from the apical area of each ear with a diameter of 8 mm (= 0.5 cm2) were excised using a disposable punch and weighed together on analytical scales.
Lymph node cell counts:
Incorporation of 3H-methyl thymidine
Both lymph nodes of one animal were prepared by gentle mechanical disaggregation through 100 mm-mesh nylon gauze with concomitant pooling in 1 mL PSB (Phosphate Buffered Saline). Cells were washed twice with an excess of PBS and precipitated with 5% trichloroacetic acid (TCA) at 4 oC 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 β-scintillation counting (Beckmann LS 6500) as disintegrations per minute (DPM)/mouse.
Positive control substance(s):
other: dinitrochlorbenzene
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 was 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:
Examination of cell proliferation in lymph nodes: in the positive control group, the SI was ≥ 3 (9.84) – test LLNA was efficient.
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 Remark
Remarks:
The SI for the test groups treated with the test substance was decreased in dose-related manner. At the highest dose level, the SI was 0.84, at the middle dose level the SI was 0.58, and at the lowest dose level, the mean SI was 0.35. The stimulation indexes of all test groups were below the threshold (see Table 1).
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The DPM for the test groups treated with the test substance was decreased in dose-related manner (see Table 1).

Any other information on results incl. tables

Table 1  Summary table

Group

Radioisotope incorporation
in lymph nodes

Ear weight

Mean DPM

SI

Mean (mg)

NC

1496.51

1.00

24.08

PC

14732.94

9.84+

32.42

30%

1249.85

0.84

24.08

3%

865.65

0.58

23.22

0.3%

522.52

0.35

23.16

Notes:

Bold 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:
Under the given test conditions, the test substance, Ashes (residues) - Biomass Combustion, elicited negative response in LLNA test.
Executive summary:

The test substance, Ashes (residues) - Biomass Combustion, 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 the test 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 Ashes (residues) - Biomass Combustion: 30%, 3%, 0.3% (w/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 9.84, which was consistent with its expected mode of action as a contact allergen.     

       The comparison of the Stimulation Indexes between the treated groups and the control group revealed that the test substance Ashes (residues) – Biomass Combustion did not cause a significant increase in radioisotope incorporation into the DNA of proliferating lymphocytes.

In conclusion, at the given experimental conditions the result of LLNA study with test substance Ashes (residues) - Biomass Combustion was negative.