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Toxicological information

Skin sensitisation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Experimental Toxicology and Ecology, BASF SE, Germany
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: solid/violet to dark blue
- Analytical purity: 96.84 %
- Expiration date of the lot/batch: 04. Feb. 2021
- Stability: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor. The stability of the test substance in drinking water at room temperature over a period of 7 days and the homogenous distribution of the test substance in drinking water was confirmed by analysis.
- Storage condition of test material: room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 19 - 22.7 g
- Housing: single housing in Makrolon cage, type II
- Diet: Kliba-Labordiaet (Maus / Ratte Haltung "GLP"), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water: Tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
5, 10, and 25%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test-substance concentration which can be technically used was a 25% test-substance preparation. The test-substance preparations at different concentrations were formulated in propylene glycol.
- Irritation: To determine the highest test-substance concentration that does not induce local signs of skin irritation and/or systemic toxicity, a pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Two mice were treated with a 25% test-substance concentration on three consecutive days. In the pre-test clinical signs were recorded after each application as well as on day 5. Signs of local irritation were recorded on day 1, 2 and 5. Furthermore, the ears were punched after sacrifice at the apical area using a biopsy punch (Ø 0.8 cm) and were immediately pooled per animal and weighed using an analytical balance. Additionally the weight of the pooled lymph nodes from both sides was determined for each animal. At the tested concentration of 25% the animals showed some increases in ear weights and lymph node weights. Moreover compound residues on the application area and blue discolored ear skin were noted during the whole observation period. Signs of systemic toxicity were not observed in the pre-test.
Therefore, the following dose levels were selected for the main study: 5%, 10% and 25%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Murine Local Lymph Node Assay
- Criteria used to consider a positive response: increase in 3H-thymidine incorporation by a factor of >/= 3, cell-count stimulation index (SI >/= 1.5), lymphnode-weight stimulation index and ear weight stimulation index together with dose-response.

TREATMENT PREPARATION AND ADMINISTRATION: The test-substance preparation was produced on a weight per weight basis shortly before the application by stirring with a high speed homogenizer (Ultra-Turrax) and/or a magnetic stirrer. The homogeneity of the test-substance preparation during application was provided by stirring with a magnetic stirrer. Propylene glycol was used as the vehicle because good homogeneity of the preparations was achieved. 25 μL per ear were applied on 3 consecutive days (day 0 – day 2) to the same application site.
Positive control substance(s):
other: A positive control is not included in the study. A separate study with a positive control (Alpha-Hexylcinnamaldehyde, techn. 85%) is carried out in the laboratory twice a year.
Statistics:
Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The stimulation indices of 3H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group. Further statistical analyses were conducted: 3H-thymidine incorporation, cell count, lymph node weight and ear weight (WILCOXON - Test).

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.89
Variability:
vehicle: 1.00
Test group / Remarks:
5%
Remarks on result:
other: statistically significant for the value p ≤ 0.01
Parameter:
SI
Value:
1.17
Variability:
vehicle: 1.00
Test group / Remarks:
10%
Remarks on result:
other: statistically significant for the value p ≤ 0.01
Parameter:
SI
Value:
2.53
Variability:
vehicle: 1.00
Test group / Remarks:
25%
Remarks on result:
other: statistically significant for the value p ≤ 0.01
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: dpm / lymph node pair: vehicle: 347.9 5%: 658.5 10%: 407.5 25%: 879.2

Any other information on results incl. tables

Cell Count Stimulation Index

   Mean [Counts / lymph node pair]  Stimulation Index
 vehicle  8,385,600  1.00
 5%  8,708,400  1.04
 10%  8,272,800  0.99
 25% 11,271,600   1.34#

Ln-Weight Stimulation Index

   Mean [mg]  Stimulation Index
 vehicle  4.9  1.00
 5%  5.4  1.09#
 10%  5.9  1.21##
 25%  7.0  1.42##

Ear-Weight Stimualtion Index

   Mean [mg]  Stimulation Index
 vehicle 28.5  1.00
 5% 31.9   1.12##
 10% 31.8  1.12#
 25% 34.0  1.19##

# = statistically significant for the value p ≤ 0.05

## = statistically significant for the value p ≤ 0.01

A slight reduction in the mean body weight was observed in test group 3. No signs of systemic toxicity were noticed in all animals during general observation. Compound residues on the application area were noted in all animals of test groups 3 and 4. Blue stained ear skin was observed at all test substance concentrations during the whole observation period.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met