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Skin sensitisation

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Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan 23 to 29, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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
Vehicle:
other: DAE 433 - mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol
Concentration:
5%, 10%,25% in DAE433
No. of animals per dose:
5
Details on study design:
The test substance was administered in the form of suspension in DAE 433.
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.
Day 1:
Open application of 25μL (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:
The weight of animals was recorded. Injection 250 μL of phosphate-buffered saline (PBS) containing 7.26 x105 Bq of 3H-methyl thymidine into all test and control mice via the tail vein.

Positive control substance(s):
other: Dinitrochlorobenzene
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
Test group / Remarks:
Negative control
Parameter:
SI
Value:
9.67
Test group / Remarks:
Positive Controls
Parameter:
SI
Value:
1.38
Test group / Remarks:
25% group
Parameter:
SI
Value:
1.03
Test group / Remarks:
10% group
Parameter:
SI
Value:
1.01
Test group / Remarks:
5% group
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Negative Control Group = 250 Positive control Group = 2418 25% solution group = 345.89 10% solution group = 257.53 5% solution group = 252.88
Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the given test conditions, the test substance does not elicit a sensitising response in LLNA assay.
Executive summary:

The positive control substance DNCB produced positive LLNA response at an exposure level expected to give an increase in the Stimulation Index SI ≥ 3 over the negative control group, which was in congruence with the expected mode of action of a contact allergen. The positive control also elicited a reaction pattern with statistically significant increase in ear weight. The negative control did not reveal any changes.These results demonstrate that the method performed in conditions of our laboratory has sufficient reliability.

The value of SI for the test groups treated by the test substance at all three dose levels is below the threshold, stimulation index

(SI) is < 3. The comparison of the Stimulation Indexes between the treated groups and the control group revealed that the test substance did not cause a statistically significant increase in radioisotope incorporation into the DNA of dividing lymphocytes. So the result of LLNA assay is negative. 

The animals exposed to the test substance showed no significant skin reactions and clinical symptoms of intoxication throughout the experiment.

The test substance showed a tendency to increase ear weight with relation to the dose.Statistically significant increase of ear weight was recorded in animals at the highest and the middle dose level (25%, 10% test substance concentrations).

This weight increase is caused by irritation effect of the test substance. The result showed the skin irritation potential considered as positive – it means the test substance is skin irritant.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Sept 9 - Oct 2, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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
Vehicle:
other: DAE 433 - mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol
Concentration:
0.5, 5, and 50% in DAE433
No. of animals per dose:
5
Details on study design:
The test substance was administered in the form of suspension in DAE 433.
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.
Day 1:
Open application of 25μL (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:
The weight of animals was recorded. Injection 250 μL of phosphate-buffered saline (PBS) containing 7.26 x105 Bq of 3H-methyl thymidine into all test and control mice via the tail vein.

Positive control substance(s):
other: Dinitrochlorobenzene
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.
Positive control results:
The positive control substance DNCB elicited a reaction pattern with statistically significant increase in ear weight and Stimulation Index of cell proliferation 10.41, which was in congruence with his expected mode of action as a contact allergen.
Parameter:
SI
Remarks on result:
other: Negative Control Group = 1.00 Positive control Group = 10.41 50% solution group = 3.00 5% solution group = 1.29 0.5% solution group = 1.22
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Negative Control Group = 219.37 Positive control Group = 2283.84 50% solution group = 658.57 5% solution group = 283.55 0.5% solution group = 268.06
Parameter:
SI
Value:
1
Test group / Remarks:
Negative Controls
Parameter:
SI
Value:
10.41
Test group / Remarks:
Positive Controls
Parameter:
SI
Value:
3
Test group / Remarks:
50% Group
Parameter:
SI
Value:
1.29
Test group / Remarks:
5% Group
Parameter:
SI
Value:
1.22
Test group / Remarks:
0.5% Group

The animals exposed to the tested concentrations of the test substance (5%, 0.5%) showed no skin reactions and clinical symptoms of intoxication. The animals exposed by the highest dose level (50%) of the test substance showed skin reactions with well defined erythema but without any clinical symptoms of intoxication throughout the experiment.

The test substance showed a tendency to increase ear weight in dose dependent manner. Statistically significant increase of ear weight was recorded only in animals at the highest dose level (50% test substance) together with skin irritation with well defined erythema.

Interpretation of results:
ambiguous
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the given test conditions, the test substance, elicited a borderline sensitising response in the LLNA assay at a 50% concentration. Positive results in cell proliferation revealed that the test substance could be a contact allergen in mice but due to the irritation that was also observed at this concentration, the possibility that it could be false positive result could not be ruled out .
Executive summary:

The positive control substance DNCB produced a positive LLNA response (SI = 10.41) at an exposure level expected to give an increase in the Stimulation Index SI ≥ 3 over the negative control group, which was in congruence with the expected mode of action of a contact allergen. The positive control also elicited a reaction pattern with statistically significant increase in ear weight. The negative control did not reveal any changes. These results demonstrate that the method performed in conditions of the laboratory has sufficient reliability.

The animals exposed to the test substance at 5% and 0.5% showed no skin reactions or clinical symptoms of intoxication. The value of stimulation index for these groups was below the positive response threshold, (SI < 3).

The animals exposed by the highest dose level (50%) of the test substance showed skin reactions with well defined erythema but without any clinical symptoms of intoxication throughout the experiment. The test substance also showed a tendency to increase ear weight in dose dependent. Statistically significant increase of ear weight was recorded only in animals at the highest dose level (50%).

The value of stimulation index for this group was right at the positive response threshold, (SI = 3).

The increased ear weight together with well defined erythema means the test substance caused irritation of skin.

The borderline positive result in cell proliferation reveals that the test substance could be a contact allergen. However, when positive irritation effects are demonstrated simultaneously, the possibility can not be ruled out, that the evaluation based on cell proliferation could be a false positive.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The test substance showed a tendency to increase ear weight at concentrations of 10% or greater. No visible skin irritation was observed at the 25% concentration. At 50% visible skin irritation was observed. Skin irritation can interfere with obtaining an accurate sensitization response and can produce false positive responses. At the 25% concentration the SI=1.38 and was clearly negative. At 50% the SI=3, which is right at the threshold for a positive result, however due to the visible skin irritation at this concentration the cell proliferation response was considered to be a false positive.


Migrated from Short description of key information:
The test substance does not elicit sensitising response in LLNA assay.

Justification for selection of skin sensitisation endpoint:
No visible skin irritation was observed at the 25% concentration. At 50% visible skin irritation was observed.

Justification for classification or non-classification

According to the CLP rules, this material does not require classification for sensitization.