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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:
2001
Report date:
2001

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
Chemical structure
Reference substance name:
3-(2-hydroxyethyl)-p-phenylenediammonium sulphate
EC Number:
298-995-1
EC Name:
3-(2-hydroxyethyl)-p-phenylenediammonium sulphate
Cas Number:
93841-25-9
Molecular formula:
C8H12N2O.H2O4S
IUPAC Name:
3-(2-hydroxyethyl)-p-phenylenediammonium sulphate
Specific details on test material used for the study:
Trade name : Betoxol II
INCI name : Hydroxyethyl-p-phenylenediamine sulfate
Batch number 100789
Assay by NMR : 98.3%
Purity by HPLC : 99.9a/a% (254nm), 99.8a/a% (292nm)
Ash content : 0.02%
Loss on drying : 0.04%
Water content : 0.04%

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca01aHsd
Sex:
female

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
0.5, 1.0, 2.0 (% w/v)
No. of animals per dose:
Five
Details on study design:
The test item was assayed at three concentrations of 0.50, 1.0, 2.0% in the vehicle DMSO. The vehicle served as a negative control. Each mouse was treated by topical application of 25uL of the selected solution onto the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days. Five days after the first topical application treatment all mice were dosed with 20uCi 3H-methyl thymidine by intravenous injection (tail vein) of 250uL of 3H-methyl thymidine diluted to a working concentration of 80 uCi/mL. Approximately 5 hours after 3H-methyl thymidine injection all mice were sacrificed. The draining auricular lymph nodes were excised, pooled for each animal (2 lymphnodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze. After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. Washing was repeated then each pellet was resuspended in approx 3mL 5% trichloroacetic acid (TCA) at ~4 deg C overnight for precipitation of macromolecules. Each precipitate was recovered by centrifugation, resuspended in 1mL 5% TCA and transfered to scintillation vials. The 3H-methyl thymidine incorporation was measured in a beta counter and expressed as the number of disintegrations per minute (DPM). Similarly background 3H-methyl thymidine levels were also measured. Determination of radioactivity was performed for each animal.The proliferative response of lymph node cells was expressed as the ratio of 3H-methyl thymidine incorporation into lymph node cells of test group animals relative to that recorded for control group animals (stimulation index). EC3, calculated concentrations which induce stimulation indices of three, were determined by linear interpolation between two points of the stimulation index axis, one above and one below the stimulation index of three. A substance will be regarded as a sensitiser in the LLNA if at least one concentration of the test item results in a 3 fold or greater increase in 3H-methyl thymidine incorporation into lymph node cells of the lymph nodes of the test group animals relative to that recorded for the lymph nodes of the control group animals.
Positive control substance(s):
other: p-phenylenediamine

Results and discussion

Positive control results:
The stimulation index for the positive control (1.0% p-phenylenediamine) was 10.1.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
2.8
Test group / Remarks:
0.5%
Parameter:
SI
Value:
4.5
Test group / Remarks:
1.0%
Parameter:
SI
Value:
7
Test group / Remarks:
2.0%
Parameter:
EC3
Value:
0.57
Remarks on result:
other: strong skin sensitiser
Cellular proliferation data / Observations:
The test item caused reactions identified as sensitisation from a concentration of 0.57% Betoxol II where the stimulation index was equal to 3.0.

Any other information on results incl. tables

 Test item  Conc/[%] DPM   Background  DPM/node  SI
 Negative control    mean  421.4  409.2  204.6  1.0
     sd  99.1      
 Betoxol II  0.50  mean  1144  1132.3  566.2  2.8
     sd  231.8      
 Betoxol II  1.0  mean  1844  1832.5  916.1  4.5
     sd  949.5      
 Betoxol II  2.0  mean  2879  2867.2  1434  7.0
     sd  928.2      
 Positive control  1.0  mean  4129  4117.2  2059  10.1
     sd  480.5      

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Hydroxyethyl-p-phenylenediamine sulfate induced a biologically relevant immune response in local lymph nodes after dermal application to the mouse ear when dimethylsulphoxude was used as the vehicle. The EC3 value was 0.57%. The concurrent positive control showed the sensitivity of the assay. Based on these findings hydroxyethyl-p-phenylenediamine sulfate (Betoxol II) is evaluated to be a strong skin sensitiser under the described test conditions.
Executive summary:

The test item was assayed at three concentrations of 0.5%, 1.0% and 2.0% employing dimethyl sulphoxde as the vehicle. Each mouse was treated by topical application with the selected solution to the entire dorsal surface of each ear once daily over three consecutive days. Five days after the first topical application treatment all mice were injected intravenously with 3H-methyl thymidine. Approximately 5 hours after 3H-methyl thymidine injection all mice were sacrificed and the draining auricular lymph nodes were excised in order to prepare a single cell suspension of the lymph node cells. The 3H-methyl thymidine incorporation was measured using a beta counter and expressed as the number of disintegrations per minute. Determination of radioactivity was performed individually for each animal. The proliferative response of lymph node cells was calculated as the ratio of 3H-methyl thymidine incorporation into lymph node cells of test group animals relative to that recorded for control group animals. A stimulation index was calculated for each concentration. The stimulation index was determined to be 2.8, 4.5, 7.0 at concentrations of 0.5%, 1.0%, 2.0% respectively. The stimulation index for the positive control (1.0% p-phenylenediamine) was 10.1. The EC3 value of the test substance was 0.57%. Based on these findings hydroxyethyl-p-phenylenediamine sulfate (Betoxol II) is evaluated to be a strong skin sensitiser under the described test conditions.

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