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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
modifications as described in publications of Ulrich et al. 2001; Ehling et al. 2005; Ehling et al. 2005. Modification includes the endpoint different from that described in original guideline (non radioactive measuring of cell proliferation).
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Physical state: red powder
- Analytical purity: ca. 99%
- Impurities (identity and concentrations): benzene, 1,2-dichloro- (ca. 0.2%), water (ca. 0.8%)
- Lot/batch No.: U7006/2007
- Expiration date of the lot/batch: 02/2023
- Storage condition of test material: ambient 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: BALB/CBYJICO
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: 17.9 to 21.5 g
- Housing: in groups of maximum six in macrolon cages
- 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: 6 days

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

Study design: in vivo (LLNA)

Vehicle:
other: DAE 433 - mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol
Concentration:
0.1%, 1%, 10%
No. of animals per dose:
6
Details on study design:
RANGE FINDING TESTS:
The highest concentration 10% (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.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA

Criteria used to consider a positive response:
The following thresholds were determined by Ulrich (2007) from analysis of historical data:
Ear weight index: 1.05; lymph node weight: 1.2; lymph node cell count: 1.3
1. Values which exceed these thresholds were considered positive
- when a statistically significant increase in one of the parameters occurs and a clear concentration-dependence can be derived
- or with no statistical significance, but a clear concentration-dependence.
2. Values which are below these thresholds were considered positive
- when a statistical significant occurs in one of the parameters together with a cIear concentration dependence.

TREATMENT PREPARATION AND ADMINISTRATION: The volume of the application form was constant in 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 (losses caused by draining from the ear must be minimized).
Positive control substance(s):
other: Dinitrochlorbenzene (DNCB)
Statistics:
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:
The positive control substance DNCB elicited a reaction pattern with statistically significant increase in ear weight and lymph node hyperplasia, which was in congruence with his expected mode of action as a contact allergen.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 1.05 (0.1%), 1.02 (1%) and 1.22 (10%)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: not applicable

Any other information on results incl. tables

The animals exposed to test substance 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 all groups in comparison to the vehicle control. There were no clinical observations attributable to the treatment with test substance.

The test substance showed a tendency to increase ear weight at the highest dose level but not paired with lymph node hyperplasia. Residues of the test substance on the ears caused this increased weight.

Summary of results

Group  LN weight  LN cell count Ear weight
Mean (mg) Index Mean (10e6/mL) Index Mean (mg) Index
NC 4.60 1.00 7.03 1.00 22.90 1.00
PC 14.78* 3.21* 29.70* 4.22* 27.55* 1.20*
0.1% 4.85 1.05 5.87 0.83 23.38 1.02
1% 4.68 1.02 6.42 0.91 23.80 1.04
10% 5.60 1.22 * 8.87 1.26 27.75* 1.21*
Figures with asterisk = values statistically significant on probability level 0.05 (Mann-Whitney test)
Figures with cross = values exceeding thresholds
NC - Negative control group
PC - Positive control group

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU