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Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

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:
other: Study performed according to OECD guideline 429 in compliance to GLP.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004
Reference Type:
other: Published secondary source
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
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)

Test material

Constituent 1
Reference substance name:
Multi constituent substance
EC Number:
916-603-5
IUPAC Name:
Multi constituent substance
Details on test material:
Test substance : Basic Red 76 (COLIPA number C008)
Batch number : 0050644101
Methylsulphate anion : 11.8%
Chloride ion : 1.6%
Water : 5.1%
o-Anisidine : 5ppm
Purity : 98.1% (HPLC)

In vivo test system

Study design: in vivo (LLNA)

Vehicle:
other: ethanol : water (7 : 3 v/v)
Concentration:
The test item was prepared in the vehicle at the following concentration : 2.5, 5.0, 10.0%
No. of animals per dose:
Four females per dose
Details on study design:
A dilution of the test item in a mixture of ethanol : water (7 : 3 v/v) was performed shortly before each dosing. A mixture of ethanol : water (7 : 3 v/v) was selected as the vehicle as a result of assessments performed on the solubility of the test item. The highest non-irritating, technically applicable test item concentration was determined in a pre-test with four mice. Based on the assessment concentrations of 2.5, 5.0, 10.0% were selected for the main study. Each test group of mice was treated by topical application to the dorsal surface of each ear lobe with the different test item concentrations. A volume of 25uL was spread over the entire dorsal surface of each ear lobe once daily for 3 consecutive days. The control group was treated with the vehicle alone. Five days after the first topical administration all mice were treated with radio labelled thymidine by intravenous injection via the tail vien. Approximately 5 hours after administration with radio labelled thymidine all mice were euthanised. The draining lymph nodes were excised and pooled for each experimental group. After preparation the level of radio labelled incorporation was then measured by scintillation counting. The proliferative response of lymph node cells is expressed as the ratio of incorporation into lymph node cells of treated animals relative to that recorded in the control animals (stimulation index). A test item is regarded as a sensitiser if the exposure to at least one concentration resulted in at least a 3 fold increase in the incorporation of radio labelled thymidine compared with concurrent controls as indicated by the stimulation index together with consideration of dose response.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
Stimulation indices of 1.5, 2.3 and 8.4 were determined for the positive control substance alpha-hexylcinnamaldehyde at concentrations of 5.0, 10.0 and 25.0% (w/v) respectively in acetone : olive oil (4:1)(v/v). The positive control item was found to be a skin sensitiser and an EC3 value of 11.7% (w/v) was derived.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
0.9
Test group / Remarks:
Group 2
Remarks on result:
other:
Remarks:
Test item concentration : 2.5%
Parameter:
SI
Value:
1.1
Test group / Remarks:
Group
Remarks on result:
other:
Remarks:
Test item concentration : 5.0%
Parameter:
SI
Value:
1.3
Test group / Remarks:
Group 4
Remarks on result:
other:
Remarks:
Test item concentration : 10.0%
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The stimulation index was 0.9, 1.1, 1.3 for test item concentrations of 2.5, 5.0, 10.0% (w/v) respectively. The calculation of the EC3 value was not performed since no test concentration produced a stimulation index of 3 or above. The positive control induced a distinct increase of the stimulation index and gave an EC3 of 11.7% (w/v).
Cellular proliferation data / Observations:
EC3 Calculation
Calculation of the EC 3 value was not performed because no test concentration produced a stimulation index of 3 or higher.

Viability/Mortality
No deaths occurred during the study period.

Clinical Signs
No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

Body Weights
The body weight of the animals, recorded on the test day 1 (prior to the first application) and on the test day 6, was within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

 Test item

concentration

/%(w/v)

 

 Measurement

/DPM

DPM-BG (a)   Number of lymph nodes

 DPM per

lymph node (b)

 Stimulation index
 -  BG 1  0  -  -  -  -
 -  BG 2  3  -  -  -  -
 -  CG 1  2297  2295  8  287  -
 2.5  TG 2  2088  2086  8  261  0.9
 5.0 TG 3   2530  2528  8  316  1.1
 10.0  TG 4  3059  3057  8  382  1.3

BG : Background (1mL 5% trichloroacetic acid) in duplicate

CG : Control group

TG : Test group

(a) : The mean value was taken from BG 1 and BG 2

(b) : Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item is not a skin sensitiser.
Executive summary:

In order to study a possible contact allergenic potential of the test item, three groups each of four female mice were treated daily with the test item at concentrations of 2.5%, 5.0% and 10.0% (w/v) in ethanol : water (7:3)(v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. The highest technically applicable concentration in the vehicle was 10.0%. A control group of four mice was treated with the vehicle (ethanol : water (7:3)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine. After approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a beta-scintillation counter. All treated animals survived the scheduled study period. No clinical signs were observed. The stimulation index was 0.9, 1.1, 1.3 for test concentrations of 2.5, 5.0, 10.0 %(w/v) respectively. A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentrations resulted in a 3 fold or greater increase in incorporation of 3H-methyl thymidine compared with concurrent controls as indicated by the stimulation index. The test item was found to be a non-sensitiser when tested up to the highest applicable concentration of 10% (w/v) in ethanol : water (7:3(v/v)).