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Toxicological information

Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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 Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Details on test material:
- Name of test material: Fluidized Bed Combustion (FBC) Bottom Ash
- Substance type: technical product
- Physical state: solid
- Appearance: Light grey solid powder with some small black particle
- Chemical structure: Complex product of oxides
- Main components: SiO2 - 27,78%, Fe2O3 - 5,98%, CaO (total) - 35,65%, Na2O - 0,25%, P2O5 - 0,38%, CO2 - 0,5%, CaO (free) - 19,91%, Al2O3 - 11,16%, TiO2 - 2,65%, MgO - 0,44%, K2O - 0,42%, SO3 (sulphate) - 2,84%
- Impurities: Metals: As, Be, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Sb, Se, Tl, V, Zn - sum < 0,1%
- Lot/batch No.: FBC/230309/T2
- Expiration date of the lot/batch: 03/2024
- Stability under test conditions: stable
- Storage: The substance will be stored in PE container at room temperature.

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760118
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 19.5-22.4 g
- Housing: Animals in groups of maximum six 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 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 appropriate SOP No.10

- Temperature (°C): 22 +/- 3oC, permanently monitored
- Humidity (%): 30 – 70 %, permanently monitored
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle: 6am-6pm/6pm-6am

Animal arrival/ start of acclimatization: 22.07.2009
Pilot experiment: 27.- 30.07 2009

Main study:
First day of administration: 05.08.2009
End of treatment period: 07.08.2009
Application of radionuclide and necropsy: 10.08.2009

Study design: in vivo (LLNA)

other: DAE 433 - mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol
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 each concentration)
Details on study design:
The highest concentration 30% (maximum technically practicable concentration) 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.

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.

Dosage volume: 25μL / ear / animal
Preparation for administration: All suspensions were prepared by suspending an appropriate amount of Fluidized Bed Combustion (FBC) Bottom Ash in the vehicle to obtain a 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: Each day of administration
Application: The volume of the application form was constant at all groups of animals - 25 l 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. Losses caused by draining from the ear must be minimized. This route of administration is listed in the guideline and it is similar to expected exposure conditions at the workplace. The application form of test substance was prepared immediately before administration.

During working hours the animals were checked for general health whenever other activities were performed – at least twice daily during the application period.

Clinical observations
Clinical signs were assessed using a defined scoring system described in SOP M/8 – at least 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 radionuclide.

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 cm2) were excised using a disposable punch and weighed together on analytical scales.

Incorporation of 3H-methyl thymidine
Both of the lymph nodes were prepared by gentle mechanical disaggregation through 100 m-mesh nylon gauze with pooling of 1 mL PSB (Phosphate Buffered Saline). Cells were washed twice an excess of PBS and precipited with 5% trichloroacetic acid (TCA) at 4 oC for 18h. 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).

Mean values and standard deviation 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. Results (incorporation of 3H-methyl thymidine) for each treatment group were expressed as the mean SI. The Stimulation Index was the ration of the mean dpm/mouse within each test substance treatment group against 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
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:
All animals in the positive control group showed these symptoms: hyperaemia of skin and clonospasm.
In the positive control group, the SI was ≥ 3 (12.31) – test LLNA was efficient.
In the positive control group, the weight of ear target was statistically significantly increased against negative control group – the test design used is efficient in the detection of irritation effect.

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: The SI for the test groups treated by the test substance was not increased with dependence on the dose level. At the all dose levels the SI was belong threshold.
other: disintegrations per minute (DPM)
Remarks on result:
other: See table No.6

Any other information on results incl. tables

Table 6.  Summary of results


Radioisotope incorporation

Ear weight

Mean DPM


Mean (mg)





















* 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
Migrated information
Under the given test conditions, the test substance, Fluidized Bed Combustion (FBC) Bottom Ash, elicited negative response in LLNA test.
Executive summary:

The test substance, Fluidized Bed Combustion (FBC) Bottom Ash, 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 Fluidized Bed Combustion (FBC) Bottom Ash 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 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 potential false positive findings with certain skin irritants.

Concentrations: positive control DNCB (dinitrochlorobenzene): 0.5% (w/v) and Fluidized Bed Combustion (FBC) Bottom Ash: 30%, 3%, 0.3% (w/v) in DAE 433.

The animals exposed to the test substance at the all dose levels 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 treated 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 12.31, which was in congruence with his expected mode of action as a contact allergen.

The test substance Fluidized Bed Combustion (FBC) Bottom Ash showed a tendency to increase ear weight with concentration-dependence, but actual increase of ear weight was not statistically significantly increased. Therefore the skin irritation effect was not considered as positive.

Comparison of Stimulation Indexes between treated groups and control group revealed that the test substance Fluidized Bed Combustion (FBC) Bottom Ash did not cause significant increase in radioisotope incorporation into the DNA of dividing lymphocytes.

The test substance Fluidized Bed Combustion (FBC) Bottom Ash had a negative result in LLNA test. Negative results in cell proliferation and no clinical symptoms of systemic toxicity revealed that the test substance Fluidized Bed Combustion (FBC) Bottom Ash is not a contact allergen in mice.

Based on the negative results of LLNA study of the test substance Fluidized Bed Combustion (FBC) Bottom Ash, it could be suggested, that the test substance, does not have to be classified as the substance, which may cause sensitisation by skin contact.