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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable, well-documented publication which meets basic scientific principles

Data source

Reference
Reference Type:
publication
Title:
Ranking of allergic potency of rubber chemicals in a modified local lymph node assay
Author:
De Jong, W., H.; et al.
Year:
2002
Bibliographic source:
Toxicological Sciences 66, 226-232

Materials and methods

Principles of method if other than guideline:
other: Local lymph node assay (using ex vivo labeling of proliferating lymph node cells, according to Kimber and Weisberger 1989 Arch. Toxicol. 63, 274-282, Vanebriel et al 2000 Toxicol. Appl. Pharmacol 162, 77-85, Van Och et al. 2000 Toxicology 146, 49-59
a modified LLNA (sodium dodecyl sulfate pretreatment and ex vivo labeling) was used
GLP compliance:
no
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
ZMBT technical grade

In vivo test system

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Central Animal Laboratory of the Institute
- Age at study initiation:
6 - 8 weeks
- Housing:
barrier-maintained under conventional conditions
- Diet (e.g. ad libitum):
yes
- Water (e.g. ad libitum):
yes

ENVIRONMENTAL CONDITIONS
in humidity, light and temperature controlled rooms

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 1.25, 2.5 5.0, 10, 20 %
No. of animals per dose:
3 per dose group, control 6
Details on study design:
Experimental design:
The sensitzing potential of the test substance was investigated in a modified local lymph node assay using ex vivo labeling of the proliferating lymph node cells (Kimber and Weisberger 1989, Vandebriel et al. 2000, Van Och et al. 2000). One hour before the chemicals were applied, 25 µl of 1% sodium dodecyl sulfate was applied epicutaneously to the dorsum of both ears to enhance response to weak sensitizer. Additionally, 25 µl of test solution or vehicle control was applied to the dorsum of both ears (50 µl per animal) of female BALB/c mice for 3 consecutive days (day 0,1, 2). At day 5 following start treatment, animals were sacrificed and draining (auricular) lymph nodes (LN) were excised. Isolated left and right LNs from each mouse were weighed and single-cell suspensions prepared using a cell strainer. Cells were washed twice and suspended in RPMI 1640 culture medium supplemented with 10% heat inactivated fetal calf serum, 100 IU(ml penicillin and 100 µg/ml streptomycin, referred to supplemented medium. Cells were counted in a Coulter Counter and adjusted to a concentration of 1x 107 cells/ml. When necessary, cell suspensions of several animals were pooled in order to obtain cell concentrations of 1 x 107 cells/ml, notably so for vehicle (AOO)-treated controls.

Lymphocyte stimulation test:
LN cell suspensions 2 x 106 cells in 200 µl were cultured in RPMI 1640 -supplemented medium in triplicate in round-bottomed 96 -well microtiter plates. An aliquot of 10 µl of 3H-methylthymidine (3H-TdR, 37kBq/ml, 100 µCi/ml) specific activity 185 GBq/mmol (5Ci/mmol in 217.8 µg/ml cold thymidine in PBS) was added to the wells of the cell culture directly after initiation of culture. Cultures were maintained for 24 h at 37°C in a humidified atmosphere of 5% CO2 in air. The cellular DNA was harvested on glass fiber filters using an automatic harvester, scintillation liquid was added, and incorporation of 3H-TdR into the DNA was measured by liquid scintillation in a ß plate counter. Proliferation per animal was determined by calculating the 3H-TdR incorporation for the total cell number harvested (left and right lymph nodes combined).
Statistics:
The EC3 (effective concentration inducing a 3 -fold increase in 3H-thymidine incorporation in the harvested lymph node cells of treated animals compared to vehicle-treated animals) was estimated by the benchmark approach, by fitting a nonlinear regression model to the data of all individual animals. The EC3 value was derivated applying likelihood/ratio tests.
In addition for estimate of uncertainty (90% confidence interval) bootstrap method was used, in addition Monte Carlo sampling was used.

Results and discussion

Positive control results:
positive control zinc diethyldithiocarbamate (ZDEC) showed an EC3 of 0.4% (0.1 to 0.8%)

In vivo (LLNA)

Resultsopen allclose all
Parameter:
EC3
Remarks:
in % concentration
Value:
30.3
Variability:
L05 - L95: 22.2 - 49.0
Test group / Remarks:
ZMBT: calclulated from test item groups; SI3 was not reached with the concentrations investigated
Remarks on result:
other: EC3 is given as effective concentration inducing a stimulation index of 3 (calculated as 30.3%); highest concentration tested was 20% for ZMBT
Parameter:
SI
Remarks:
given in cpm/animal
Value:
2 056
Variability:
+/- 176
Test group / Remarks:
5% ZMBT
Parameter:
SI
Remarks:
given in cpm/animal
Value:
1 580
Variability:
+/- 99
Test group / Remarks:
10% ZMBT
Parameter:
SI
Remarks:
given in cpm/animal
Value:
2 659
Variability:
+/- 529
Test group / Remarks:
20% ZMBT
Parameter:
EC3
Remarks:
in % concentration
Value:
0.4
Variability:
L05 - L95: 0.2 - 0.6
Test group / Remarks:
positive control zinc diethyldithiocaramate (ZDEC)
Remarks on result:
other: EC3 is given as effective concentration inducing a stimulation index of 3 (determined with 0.4%)

Any other information on results incl. tables

ZMBT: EC3*: 30.3 % (L05 -L95: 22.2 -49 -0, estimated 90% confidence interval based on 200 bootstrap runs)

*effective concentration inducing a stimulation index of 3 in LLNA

Moderate skin sensitzing

Table: Data dose response study with ZMBT

 ZMBT concentration (%)  LN weight (mg)  LN cell no x 106  Proliferation (cpm/2 x 106) LN cells  LN proliferation (cpm/animal)
 0  3.2 ± 0.2 (12)  3.6±0.2 (6) 638  (2)  1144±74 (6)
1.25  3.0± 0.2 (6)  3.8±0.6  655 (1)  1244±180
 2.5  3.8± 0.2 (6)  4.1±0.9  799 (1)  1638±346
 5.0  3.9 ± 0.2 (6) 3.8 ±0.3 1073 (1)  2056±176
 10.0 3.8  ± 0.2 (6)  3.7±0.2  846 (1)  1580±99
 20.0  4.4± 0.2 (6) 5.3 ±1 -0  1010 (1)  2659±529

note: Arithmetic means and standard errors of the mean (SEM); n=3 unless stated otherwise (in parentheses)

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Remarks:
Migrated information
Executive summary:

The skin sensitizing potential of ZMBT was evaluated in an acceptable documented modified Local Lymph Node Assay (LLNA) (De Jong 2002). A modified LLNA with ex vivo tritium thymidine (3H-TdR) labelling of the proliferating lymph node cells was used for the evaluation. Young adult female BALB/c mice (3 per dose group) were used in this modified LLNA; and were treated with 0, 1.25, 2.5, 5.0, 10.0 and 20 % test substance solution daily for three consecutive days (day 0, 1 and 2). A concurrent solvent control (AOO) was also included (6 animals). At day 5 following start of treatment animals were sacrificed and draining lymph nodes were excised. The local lymph nodes were weighed and single cell suspensions were prepared. These cells were cultured in presence of tritium thymidine (3H-TdR) to determine cell proliferation of the prepared lymph nodes per animal. The EC3 (effective concentration inducing a 3-fold increase in proliferation of lymph node cells) was calculated with non-linear regression analysis. For the ZMBT an EC3 value of 30.3% was calculated by extrapolation of the experimental results. Based on this calculation a moderate skin sensitizing potential of ZMBT is suggested.