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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2.2.2010-16.2.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
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

Constituent 1
Reference substance name:
Slimes and Sludges, blast furnace and steelmaking
EC Number:
266-006-2
EC Name:
Slimes and Sludges, blast furnace and steelmaking
Cas Number:
65996-73-8
Molecular formula:
not applicable
IUPAC Name:
Sludges steelmaking
Details on test material:
- Physical state: solid
- Composition of test material, percentage of components: Fe (total) 54.29% (mainly as oxides), CaO 10.17%, Zn 5.25%, MgO 4.32%, C 2.06%, SiO2 1.64%, Mn 0.69%, K2O 0.126%, Na2O 0.239%, Al2O3 0.16%
- Lot/batch No.: 21.10.2009
- Expiration date of the lot/batch: unlimited
- Stability under test conditions: unlimited
- Storage condition of test material: stored in PE container 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., Kolec u Kladna, Czech Republic
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 19.4-22.6 g
- Housing: group-wise five in macrolon cageswith sterilized softwood shavings, cleaning and disinfection of animal room was regularly performed (according to SOP No. 10)
- Diet (e.g. ad libitum): pelleted standard diet for experimental animals ad libitum
- Water (e.g. ad libitum): drinking tap water ad libitum (water quality corresponded to Ministerial Decree No. 252/2004 Czech Coll of Law)
- Acclimation period: 5 days at least


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C (permanently monitored)
- Humidity (%): 30-70 % (permanently monitored)
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Study design: in vivo (LLNA)

Vehicle:
other: DAE 433: dimethylacetamide (40 %), acetone (30 %), ethanol (30 %)
Concentration:
30% (w/v) (300 mg/ml)
3% (w/v) (30 mg/ml)
0.3% (w/v) (3 mg/ml)
No. of animals per dose:
5 animals/dose
Details on study design:
MAIN STUDY
Experimental schedule:
Day 1:
Open application of 25 µl (in the morning, by pipette) of appropriate emulsions of the test substance, the vehicle alone or the positive control to the dorsum of each ear.
Days 2 and 3:
The application procedure repeated as carried out on day 1.
Days 4 and 5:
No treatment.
Day 6:
Injection of 250 µl of phosphate-buffered saline (PBS) containing 7.1 x105 Bq of 3H-methyl thymidine into all test and control mice via the tail vein. Five hours later, the animals were killed.


PILOT TEST
The highest test-substance concentration of 30% was administered to three animals to assess possible systemic toxicity. During the pilot experiment clinical symptoms of systemic toxicity were not observed. No macroscopic changes (after necropsy) were found in all three animals.


TREATMENT PREPARATION AND ADMINISTRATION:
Application:
The volume of the dose was constant for 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 to avoid losses caused by draining from the ear.

Preparation for administration: All emulsions were prepared by suspending an appropriate amount of Slimes and Sludges, blast furnace and steelmaking in the vehicle to obtain a concentration of 30%, 3% or 0.3% (w/v). The emulsions were prepared before the start of application by mixing on magnetic stirrer and were still being mixed during application.

Frequency of preparation: Each day of administration
Positive control substance(s):
other: 1-chloro-2,4-dinitrobenzene
Statistics:
For statistical calculations the software Statgraphic(R) 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:
see Table 1

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.52
Test group / Remarks:
0.3 %
Key result
Parameter:
SI
Value:
1.29
Test group / Remarks:
3 %
Key result
Parameter:
SI
Value:
1.59
Test group / Remarks:
30 %

Any other information on results incl. tables

Table 1: Cell suspensions radioactivity

Group Radioisotope incorporation in lymph nodes
Mean DPM stimulation index
negative control 237.26 1.00
positive control 4018.42 16.94
30% 377.19 1.59
3% 305.17 1.29
0.3% 359.65 1.52

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test substance Slimes and Sludges, blast furnace and steelmaking had a negative result in LLNA test.
Executive summary:

The test substance, Slimes and Sludges, blast furnace and steelmaking, was tested for the assessment ofskin 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 radionuclide 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 Slimes and Sludges, blast furnace and steelmaking was evaluated after topical application to female BALB/c mice. Five mice per group were exposed on the dorsum of both ears by test and control substances 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.

Concentrations: positive control DNCB (2,4-dinitrochlorobenzene): 0.5% (w/v) and Slimes and Sludges, blast furnace and steelmaking: 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 clinical symptoms of intoxication throughout the experiment were observed at the all dose levels. 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 cell proliferation, the Stimulation Index reaching 16.94, 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 Slimes and Sludges, blast furnace and steelmaking did not cause a significant increase in radioisotope incorporation into the DNA of dividing lymphocytes. This effect was dose dependent. The maximal SI was 1.59 at the highest test concentration of 30 %.

The test substance Slimes and Sludges, blast furnace and steelmaking had anegative result in LLNA test.

Based on these results the test substance, Slimes and Sludges, blast furnace and steelmaking, does not have to be classified as the substance, which may cause sensitisation by skin contact.