Benzimidazole-2-thiol

Endpoint summary

Description of key information

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.

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.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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

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)

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:

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.

Key result

Parameter:

EC3

Value:

14.7

Test group / Remarks:

test group

Remarks on result:

other: positive sensitizer

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Various studies have been reviewed to determine the allergenic potential of the test chemical in living organisms. These include in vivo experimental studies performed on mice, humans, guinea pigs.

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.

This is supported by the results of Freunds Complete Adjuvant test performed to evaluate the dermal sensitization potential of the test chemical.

The Technical-grade test chemical was tested in male albino guinea pigs with regard to their sensitizing potential. Induction was carried out in different ways in 4 series of test. Induction was carried out in different ways in 4 series of test.

In test series I, groups of 36 guinea pigs received daily epicutaneous applications of 0.03 ml of a 3-percent acetone solution of test chemical for a period of 3 weeks. The total dose of test chemical applied per animal was 18.9 mg. The animals in test series II (40 guinea pigs per group) were given intradermal injections of 0.1 ml heparin and subsequently, on 8 days within a period of 3 weeks, they each received epicutaneous applications of 0.03 ml of a 5-percent acetone solution of test chemical in the area around the heparin injection site. The total dose of test chemical administered per animal was 12 mg in both cases. The guinea pigs in test series III (groups of 40 animals) were given intradermal injections of 1000 μg test chemical in 0.01 ml Freund’s adjuvant into the ear. In test series IV, 3 guinea pigs received daily epicutaneous 0.03 ml applications of a 3% acetone solution of test chemical respectively, plus intradermal injections of 100 μg test chemical respectively, as solutions in 0.01 ml Freund’s adjuvant for 3 weeks. The total dose of test chemical which was administered per animal was 18.3 mg. Each test series included a control group of 25 animals which was treated with the solvent in the appropriate manner. Eleven to 14 days after the end of the induction phase, the guinea pigs underwent epicutaneous challenge with 750 μg test chemical which was applied to the untreated skin of the flank. In all 4 test series, the animals treated with test chemical exhibited sensitisation reactions.

Thus from the above study it was concluded that technical-grade test chemical was considered to be a weak skin sensitising agent.

In a modified maximisation test carried out in guinea pigs, the test chemical dissolved in olive oil as the vehicle, was tested for its sensitising potential at concentration levels of 0.5 and 5%. The first induction was carried out intradermally with 0.5 and 5% test chemical both with and without emulsified Freund’s adjuvant. In addition to a solvent control, the study included a positive control with 2,4-dinitrochlorobenzene (0.1%). The second induction was performed dermally using a 20-percent formulation of the test chemical. The challenge was carried out with the test chemical at concentration levels of 0.05, 0.5 and 5%. Under these experimental conditions, the test chemical did not cause any sensitizing reactions in guinea pigs. Similarly, the skin of guinea pigs showed no cross reactions to chemically related products.

The above result is also supported by a patch test performed to evaluate the dermal reactions caused by the test chemical. 17 patients allergic to Mercaptobenzothiazole, were tested with the test chemical and other rubber accelarators. The test chemical 1% in petrolatum to the skin of 17 volunteers and reactions were read at 48 hours. The tests were negative with the 20 controls tested.

The test chemical caused sensitizing reactions in 2 of the 17 patients tested with 1% test chemical in petrolatum.

Hence, the test chemical was considered to be not sensitizing to the skin of humans.

The local lymph node assay (LLNA) was performed to determine the allergenic potential of the test chemical. The test was performed according to OECD 429 Guidelines.

EC3 value is the estimated concentration of a chemical necessary to give a 3-fold stimulation of proliferation in lymph nodes compared to concurrent vehicle-treated controls. The vehicle for these studies was acetone:olive oil, 4 : 1, v ⁄ v.

The EC3 value of the test chemical was 14. It was considered to be moderate sensitizer to skin.

Even though the results of the maximization test and patch test suggest that the test chemical may not cause any sensitization to skin, but the results of LLNA studies indicate otherwise. Taking into considerations all this parameters, the test chemical can be considered to be sensitizing to skin.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Even though the results of the maximization test and patch test suggest that the test chemical may not cause any sensitization to skin, but the results of LLNA studies indicate otherwise. Taking into considerations all this parameters, the test chemical can be considered to be sensitizing to skin.