Benzimidazole-2-thiol

Skin sensitisation

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:

data from handbook or collection of data

Justification for type of information:

data is from peer reviewed journals

Data source

Reference

Reference Type:

publication

Title:

Ranking of Allergenic Potency of Rubber Chemicals in a Modified Local Lymph Node Assay

Author:

Wim H. De Jong, Francois M. M. Van Och, Constance F. Den Hartog Jager, Sander W. Spiekstra, W. Slob, Rob J. Vandebriel, and Henk Van Loveren

Year:

2002

Bibliographic source:

TOXICOLOGICAL SCIENCES, 66, 226–232, (2002)

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Modified Local Lymph Node assay

Principles of method if other than guideline:

The sensitizing potency of the chemicals was investigated in a modified local lymph node assay using ex vivo labeling of the proliferating lymph node cells

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Benzimidazole-2-thiol

EC Number:

209-502-6

EC Name:

Benzimidazole-2-thiol

Cas Number:

583-39-1

Molecular formula:

C7H6N2S

IUPAC Name:

1H-benzimidazole-2-thiol

Details on test material:

SOURCE OF TEST MATERIAL
- Test substance: 2-mercaptobenzimidazole (benzimidazole-2-thiol) (CAS No. 583-39-1) is a pale yellow white powder with a molecular weight of 150.21.
- Source of test material: Sumitomo Chemical Co., Ltd.
- Lot number: 30807
- Purity: 98.5%
- Substance type: Organic
- Physical state: Solid
- Molecular formula: C7H6N2S
- Molecular weight: 150.204 g/mol

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test substance was kept at room temperature with light shielding until use.
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: Stability tests of 2-mercaptobenzimidazole in DMSO solution were carried out at low concentration (3.125 mg / ml) in this study and high concentration (300 mg / ml) in chromosomal aberration test conducted simultaneously in our laboratory 2 concentration under light-shielded condition at room temperature. As a result, the average content of each 3 samples after preparation for 4 hours was 99.7 and 108% with respect to the average of the initial value (0 hour), respectively. In addition, as a result of conducting the content measurement test on the prepared specimens used in this Test I, the content of the 3.125 mg / ml solution was 96.2 to 97.1% with respect to the predetermined concentration, the content of the 50 mg / ml solution was 95.4 to 96.2% there were.
From the above results, it was confirmed that the 2-mercaptobenzimidazole was stable in the DMSO solution and the content of the test substance in the preparation solution was within the predetermined value range.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 2-Mercaptobenzimidazole was prepared by adjusting the concentration to 50 mg / ml in dimethylsulfoxide (hereinafter abbreviated as DMSO, Wako Pure Chemical Industries, Ltd.), then further diluting it with the same solvent at a common ratio of 2 to about 3 was used for the test promptly.

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.
- Females (if applicable) nulliparous and non-pregnant: [yes/no/not specified]
- Microbiological status of animals, when known:
- Age at study initiation:
- Weight at study initiation:
- Housing:
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period:
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):
- IN-LIFE DATES: From: To:

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

25 microliter of test solution

Details on study design:

PRE-SCREEN TESTS:
One hour before the test chemical was applied, 25 ml of 1% sodium dodecyl sulfate (SDS, Merck BV, Amsterdam, The Netherlands) was applied epicutaneously to the dorsum of both ears to enhance responses to weak sensitizers

MAIN STUDY : 25 microliter of test solution or vehicle control was applied to the dorsum of both ears (50 ml per animal) of female BALB/c mice daily for 3 consecutive days (days 0, 1, and 2). At day 5 following start of 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 (Falcon, Franklin Lakes,NJ, USA). Cells were washed twice and suspended in RPMI 1640 (Gibco, Grand Island, NY, USA) culture medium supplemented with 10% heat inactivated fetal calf serum (PAA, Linz, Austria), 100 IU/ml penicillin, and 100 mg/ml streptomycin, referred to as supplemented medium. Cells were counted in a Coulter Counter (Coulter Electronics, Mijdrecht, the Netherlands) and adjusted to a concentration of 1*107 cells/ml. When necessary, cell suspensions of several animals were pooled in order to obtain cell concentrations of 1*107 cells/ml, notably so for vehicle (AOO)-treated controls.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Modified Local Lymph Node Assay
- Criteria used to consider a positive response: The EC3 concentration (concentration of the chemical inducing an SI >3) was estimated using a benchmark approach

TREATMENT PREPARATION AND ADMINISTRATION:
Lymphnode cell suspensions, 2*106 cells in 200 ml, were cultured in RPMI 1640-supplemented medium in triplicate in round-bottomed, 96-well microtiter plates (Greiner, Alphen aan de Rijn, The
Netherlands). An aliquot of 10 ml of 3H-methylthymidine (3H-TdR, Amersham International, Buckinghamshire, UK), 37 kBq/well, 3.7 MBq/ml, 100 mCi/ml), specific activity 185 GBq/mmol (5 Ci/mmol in 217.8 mg/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 cell harvester (Harvester 96t Tomtec, Orange, CT), scintillation liquid was added, and incorporation of 3H-TdR into the DNA was measured by liquid scintillation in a b plate counter (1205 BetaplateTM, Wallac, Turku,Finland). Proliferation per animal was determined by calculating the 3H-TdR incorporation for the total cell number harvested (left and right lymph nodes combined).

Positive control substance(s):

not specified

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 choice of the model for deriving the EC3 follows from a procedure of applying likelihood ratio tests on the members of the following nested family of models.
Model 1: y= a
Model 2: y = a exp(bx)
Model 3: y = a exp(bxd)
Model 4: y = a(c – (c – 1)exp(bx))
Model 5: y = a(c – (c – 1)exp(bxd)),

where y is the response, and x denotes the applied concentration. The parameter a represents the level of response at concentration zero, and b can be considered as the parameter reflecting the efficacy of the chemical. At high doses, models 4 and 5 level off to the value ac, so the parameter c can be interpreted as the maximum relative change compared to the background. Models 3 and 5 have the flexibility to mimic threshold-like responses. All these parameters. Therefore, these 2 models cannot be (formally) compared to each other by a likelihood-ratio test. For each data set (compound), one of these models was selected by choosing a more complicated model when the increase in number of parameters resulted in a significantly better fit to the dose-response data. The selected model was used to estimate the EC3 (point estimate). Additionally, an estimate of the uncertainty (90% confidence interval) associated with the estimated EC3 was determined using a (parametric) bootstrap method, as follows. Once a model is selected for describing the dose-response data, this fitted model is used as a basis for generating 200 artificial data sets (according to the experimental design) by Monte Carlo sampling. For each generated data set, the EC3 is re-estimated. Taking all these EC3s together results in a distribution representing the uncertainty associated with the EC3 estimate.

Results and discussion

In vivo (LLNA)

Results

Key result

Parameter:

EC3

Value:

14.7

Test group / Remarks:

test group

Remarks on result:

other: positive sensitizer

Any other information on results incl. tables

CAS	EC3*	L05-L95**	LLNA Ranking***
583-39-1	14.7	11.8-19.8	10

 

*- Effective concentration inducing a stimulation index of 3 in LLNA

**- Estimated 90% confidence interval based on 200 bootstrap runs

***- NR = EC3 not reached according to fitted model

Applicant's summary and conclusion

Interpretation of results:

other: Sensitizing

Conclusions:

The EC3 value for the test chemical was 14.7. Based on this value the test chemical was considered to be moderate sensitizer to skin.

Executive summary:

The sensitizing potency of the chemicals was investigated in a modified local lymph node assay using ex vivo labeling of the proliferating lymph node cells. One hour before the test chemical was applied, 25 ml of 1% sodium dodecyl sulfate (SDS, Merck BV, Amsterdam, The Netherlands) was applied epicutaneously to the dorsum of both ears to enhance responses to weak sensitizers. 25 microliter of test solution or Acetone: olive oil[4:1] was applied to the dorsum of both ears (50 ml per animal) of female BALB/c mice daily for 3 consecutive days (days 0, 1, and 2). At day 5 following start of 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 (Falcon, Franklin Lakes,NJ, USA). Cells were washed twice and suspended in RPMI 1640 (Gibco, Grand Island, NY, USA) culture medium supplemented with 10% heat inactivated fetal calf serum (PAA, Linz, Austria), 100 microliter/ml penicillin, and 100 mg/ml streptomycin, referred to as supplemented medium. Cells were counted in a Coulter Counter (Coulter Electronics, Mijdrecht, the Netherlands) and adjusted to a concentration of 1*107 cells/ml. When necessary, cell suspensions of several animals were pooled in order to obtain cell concentrations of 1*107 cells/ml, notably so for vehicle (AOO)-treated controls.

The EC3 concentration (concentration of the chemical inducing an SI > 3) was estimated using a benchmark approach

The EC3 value for the test chemical was 14.7. Based on this value the test chemical was considered to be moderate sensitizer to skin.