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Description of key information

Information on sensitization are derived from reliable, experimental data of a strucutral analogue. The skin sensitizing potential of the test item (analogue) was assessed in a murine local lymph node assay in mice (OECD 429, GLP). 0.2, 2 and 20% solution was applied onto the dorsum of both ears for three consecutive days. The test item did not cause clinical symptoms and the SI of all treatment groups was below 3. Therefore, the substance is not considered to be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeding farm VELAZ S.LO., Kolec u Kladna, Czech Republic, RCH CZ 21760118
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 16.6 - 19.22 g
- Housing: 6/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): /
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
other: DAB 433 - mixture of40% dimethylacetamide, 30% acetone and 30% ethanol
Concentration:
0.2, 2, 20 % w/v
No. of animals per dose:
5/dose
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: 20% technically highest concentration
- Irritation: no effects or clinical symptoms
- Lymph node proliferation response:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: SI >=3

TREATMENT PREPARATION AND ADMINISTRATION: 25 ul 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.

Day 1:
Open application of 25f.lL (in the morning, by pipette) of appropriate suspensions 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 250 ul of phosphate-buffered saline (PBS) containing 7.425 x10^5 Bq of 3H-methyl thymidine into all test and control mice via the tail vein. Five hours later, the animals were killed.
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.
Parameter:
SI
Value:
1
Variability:
negative control
Parameter:
SI
Value:
9.56
Test group / Remarks:
positive control
Parameter:
SI
Value:
1.18
Test group / Remarks:
0.2 % test group
Parameter:
SI
Value:
1.05
Test group / Remarks:
2 % test group
Parameter:
SI
Value:
1.58
Test group / Remarks:
20 % test group
Parameter:
other: disintegrations per minute (DPM)
Value:
84.46
Test group / Remarks:
negative control (mean)
Parameter:
other: disintegrations per minute (DPM)
Value:
807.23
Test group / Remarks:
positive control (mean)
Parameter:
other: disintegrations per minute (DPM)
Value:
99.78
Test group / Remarks:
0.2 % test group
Parameter:
other: disintegrations per minute (DPM)
Value:
88.33
Test group / Remarks:
2 % test group
Parameter:
other: disintegrations per minute (DPM)
Value:
133.59
Test group / Remarks:
20 % test group
Interpretation of results:
GHS criteria not met
Conclusions:
Under the given test conditions, the test substance does not elicit sensitising response in LLNA assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Read across justification

The skin sensitization potential of the test item was not assessed. Reliable data of a structural analogue are available. Both substances are calcium-salts and share high similarity in structure. Moreover, the substances are minimal soluble in water. Therefore, it is acceptable to derive information on sensitization from experimental data of the analogue substance. A detailed read across justification is given in Annex I of the CSR.

Procedure and observations

The substance was tested for the assessment of skin sensitisation potential with the murine local lymph node assay with radionuclides in mice. Five mice per group were exposed on the dorsum of both ears once a day by test substance (0.2, 2, 20%) and control substances during 3 consecutive days. Primary proliferation of lymphocytes in the lymph node was evaluated and the SI determined. The animals exposed to the test substance at all concentrations showed no pathological skin reactions and no other negative clinical symptoms of intoxication. Comparison of Stimulation Indexes (SI) between all treated groups and control vehicle group revealed that the test substance did not cause significant increase in radioisotope incorporation into the DNA of dividing lymphocytes.

Discussion

Thus, the test item is not considered to be a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for skin sensitization under Directive 67/548/EEC,as amended for the 30th time in Directive 2008/58/EC.

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008, as amended for the second time in Directive (EC 286/2011).