Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
03 Jan 2008 - 17 Jan 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study. In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
Test conducted with modifications (nonradioactive method) described in Ulrich et al. (2001. Arch Toxicol 74:733-744), Ehling et al. (2005. Toxicology 212: 1-11 and 60-68). Threshold values for evaluation based on a nonpublished personal communication.
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 (as cited in study report): Ashes (residues)
- Substance type: technical product
- Physical state: solid
- Significant impurities:
Metals with content lower than 0.1%: Cr, Cu, Zn
Metals with content lower than 0.01%: As, Pb, Cd, Hg
- Composition of test material, percentage of components:
Main components: SiO2 (42.12%), Al2O3 (32.16%) and Fe2O3 (10.88%)
Components with content lower than 10%: TiO2, MnO, MgO, CaO, Na2O, K2O, SO3
- Lot/batch No.: 30.08.2007_ENERGETIKA
- Expiration date of the lot/batch: unlimited
- Stability under test conditions: stable
- Storage condition of test material: labotatory conditions
- Appearance: greyish black 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 BioTest s.r.o., Konárovice, 281 25 Czech Republic, RČH CZ 21760152
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 21-24.6 g
- Housing: groups of six animals (or less) in macrolon cages with sterilized softwood shavings
- Diet (e.g. ad libitum): Pelleted standard diet for experimental animals ad libitu
- Water (e.g. ad libitum): Drinking tap water ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): ca. 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 14 Jan 2008 To: 17 Jan 2008

Study design: in vivo (LLNA)

Vehicle:
other: DAE 433 mixture of 40% 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. Concentrations in formulations were:
30% (w/v) 300 mg/mL
3% (w/v) 30 mg/mL
0.3% (w/v) 3 mg/mL
No. of animals per dose:
Pilot experiment: 3 females
Exposed groups: 18 females (6 animals in 3 groups)
Positive control group: 6 females
Negative control group: 6 females
Reserve group: 4 females
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The test material is not soluble. Therefore, suspensions of the test material in vehicle were prepared and tested.
- Irritation: No irritation, no signs of systemic toxicity and no macroscopic changes (after necropsy) were observed in a pilot experiment conducted with 3 animals treated with a 30% suspension of the test material.
- Lymph node proliferation response: Proliferation (cell concentration in the lymph nodes) was measured by cell counting, which served as parameter for comparison between test and control groups.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response:
The results of the LLNA were evaluated according to the following criteria.
According to the authors, the following threshold values had been determined from analysis of historical data and were reported as a personal communication:
Ear weight index: 1.05
LN weight: 1.2
LN cell count: 1.3

1. Values exceeding these thresholds were considered as a positive result
- if a statistically significant increase in one of the parameters compared to control occurs and a clear concentration dependence can be derived
- or, in the absence of statistical significance, a clear concentration dependence is observed.

2. Values below these thresholds were considered as a positive result
- if one of the parameters showed a statistically significant increase compared to control, along with a clear concentration dependence.

3. Values were considered as a negative result
- in case of being below the threshold values and without a statistically significant increase compared to control
- in case of being below the threshold values, with a statistically significant increase compared to control, but without showing a clear concentration dependence
- in case of being above the thresholds, without a statistically significant increase compared to control, and without showing a clear concentration-dependence.


TREATMENT PREPARATION AND ADMINISTRATION:
- Dosage volume: 25μL/ear/animal
- Preparation of suspensions: All suspensions were prepared by mixing an appropriate amount of the test material in the vehicle in order to obtain a concentration of 30%, 3% or 0.3% (w/v). Prior to application, the suspensions were mixed for 5 minutes with a magnetic stirrer.
- Frequency of preparation: each day of application
- Application: 25 µL of the appropriate dilution was applied to the dorsum of each ear once a day for 3 consecutive days.
- Other examinations:

Ear and lymph node weights: Animals were sacrificed 24 h after the last application. Immediately after sacrifice both ears were sectioned 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 balances. Pairs of auricular LN were excised and weighed on analytical balances.

Lymph node cell counts: Both lymph nodes were prepared by gentle mechanical disaggregation through a 100-µm-mesh nylon gauze with pooling of 1 mL PSB (Phosphate Buffered Saline). The cell concentration in the resulting suspension was measured with a Counter Celltac-α (Nihon Kohden) and analysed with veterinary software.
Positive control substance(s):
other: dinitrochlorbenzene (DNCB, 0.5% w/v in vehicle)
Statistics:
For statistical calculations, the software Statgraphic ® Centurion (version XV, USA) was used. At first, the global comparison of all three values of the test groups to 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:
In the positive control group, the cell concentration in suspension and weight of lymph nodes was statistically significantly increased compared to the negative control group.
In the positive control group, the weight of the excised ear discs was statistically significantly increased compared to the negative control group.
The positive control substance DNCB elicited a reaction pattern with statistically significant increase in ear weight and LN hyperplasia, which was in congruence with the expected mode of action of a contact allergen.

Any other information on results incl. tables

No statistically significant increases in lymph node cell count as well as ear and lymph node weights were observed in any of the test groups. The index for LN cell count exceeded the threshold value in the 0.3 and 30% test groups without a clear dose response.

Summary of results

Group

LN weight

LN cell count

Ear weight

Mean (mg)

Index

Mean (106/mL)

Index

Mean (mg)

Index

NC

5.62

1.00

8.55

1.00

26.40

1.00

PC

13.52*

2.41+

26.00*

3.37+

32.37*

1.23+

0.3%

5.62

1.00

12.22

1.43+

24.83

0.94

3%

6.32

1.12

9.93

1.16

25.18

0.95

30%

6.43

1.15

11.78

1.38+

26.25

0.99

* = values statistically significant on probability level 0.05 (Mann-Whitney test)

+ = values exceeding threshold values

NC: Negative control group

PC: Positive control group

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under the conditions described in this study, the test substance elicited negative results in the LLNA.