1,3-dibutyl-2-thiourea

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

HPRT

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February - April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro gene mutation study in mammalian cells (HPRT)

Test material

Test material information

Constituent 1

Reference substance name:

1,3-dibutyl-2-thiourea

EC Number:

203-674-6

EC Name:

1,3-dibutyl-2-thiourea

Cas Number:

109-46-6

Molecular formula:

C9H20N2S

IUPAC Name:

1,3-dibutyl-2-thiourea

Test material form:

solid: particulate/powder

Details on test material:

None.

Method

Target gene:

hprt locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co.
- Storage at Covance: as frozen stocks in liquid notrogen.
Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free.
For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37+/-1°C. When the cells were growing well, subcutltures were established in an appropriate number of flasks.

MEDIA USED
- Type and identity of media: RPMI 1640 media containing L-glutamine and HEPES

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

Concentrations selected for the Mutation Experiment were based on the results of the cytotoxicity Range-Finder Experiment.

Range finder (+/-S9): 37.5-75-150-300-600-1200 µg/ml
Mutation experiment (-S9): 50-100-200-300-350-400-450-500-550-600-700 µg/ml
Mutation experiment (+S9): 25-50-100-200-300-350-400-450-500-550-600 µg/ml

Vehicle / solvent:

DMSO
Preliminary solubility data indicated that N,N’-Dibutylthiourea was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 221.1 mg/mL. The solubility limit in culture medium was in the range of 276.4 to 552.8 µg/mL, as indicated by precipitation at the higher concentration which persisted for 3 hours after test article addition, with warming at 37°C. A maximum concentration of 1200 µg/mL was therefore selected for the cytotoxicity Range-Finder.

Test article stock solutions were prepared by formulating N,N’-Dibutylthiourea under subdued lighting in DMSO, with the aid of vortex mixing and ultrasonication, to give the maximum required concentration. Subsequent dilutions were made using DMSO.The test article solutions were protected from light and used within approximately 1.5 hours of initial formulation.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
At least 107 cells in a volume of 18.8 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 0.2 mL vehicle, test article, or positive control solution was added. S-9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.

DURATION
- Preincubation period: 3h
- Exposure duration: 7d
- Expression time (cells in growth medium): 7d

NUMBER OF CELLS EVALUATED: At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 105 cells/mL in readiness for plating for 6TG resistance.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: Cloning Efficiency (CE) in any given culture is therefore: CE = P/No of cells plated per well, and as an average of 1.6 cells/well were plated on all survival and viability plates, CE = P/1.6.
Percentage Relative Survival (% RS) in each test culture was determined by comparing plating efficiencies in test and control cultures thus: % RS = [CE (test)/CE (control)] x 100.
To take into account any loss of cells during the 3 hour treatment period, percentage relative survival values for each concentration of test article were adjusted as follows: Adjusted % RS = [% RS x Post-treatment cell concentration for test article treatment] / Post-treatment cell concentration for vehicle control


- OTHER: metabolic activation system
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it is prepared from male Sprague Dawley rats induced with Aroclor 1254. The batches of S-9 were stored frozen in aliquots at <-50°C prior to use (Booth et al., 1980). Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufin-O-dealkylase activities).
The S-9 mix was prepared in the following way: G6P (180 mg/mL), NADP (25 mg/mL), KCl (150 mM) and rat liver S-9 were mixed in the ratio 1:1:1:2. For all cultures treated in the presence of S-9, an aliquot of the mix was added to each cell culture to achieve the required final concentration of test article in a total of 20 mL. The final concentration of the liver homogenate in the test system was 2%.

Rationale for test conditions:

Acceptance Criteria: The assay was considered valid if the following criteria were met:
1. The MF in the concurrent negative control was considered acceptable for addition to the laboratory historical negative control database,
2. The MF in the concurrent positive controls induced responses that were compatible with those generated in the historical positive control database and give a clear, unequivocal increase in MF over the concurrent negative control,
3. The test was performed with and without metabolic activation,
4. Adequate numbers of cells and concentrations were analysable.

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The MF at one or more concentrations was significantly greater than that of the vehicle control (p=0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p=0.05)
3. The results were outside the historical vehicle control range.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological relevance. Extreme caution was exercised with positive results obtained at levels of RS lower than 10%.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Cytotoxicity
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 37.50 to 1200 µg/mL (a precipitating treatment concentration). Upon addition of the test article to the cultures, precipitate was observed at the highest three concentrations in the absence and presence of S-9 (300 to 1200 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the top two concentrations in the absence and presence of S-9 (600 and 1200 µg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S-9 was retained and the higher concentration discarded. The highest concentrations to give >10% RS were 600 µg/mL in the absence of S-9 and 300 µg/mL in the presence of S-9, which gave 11% and 39% RS, respectively.
No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentrations analysed (600 µg/mL) as compared to the concurrent vehicle controls

In the Mutation Experiment eleven concentrations, ranging from 50 to 700 µg/mL in the absence of S-9 and from 25 to 600 µg/mL in the presence of S-9, were tested.
Upon addition of the test article to the cultures, precipitate was observed at the highest six concentrations in the absence of S-9 (400 to 700 µg/mL) and the highest five concentrations in the presence of S-9 (400 to 600 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest five concentrations in the absence of S-9 (450 to 700 µg/mL) and the top three concentrations in the presence of S-9 (500 to 600 µg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S-9 was retained and higher concentrations were discarded. Seven days after treatment all remaining concentrations in the absence of S-9 were selected to determine viability and 6TG resistance. The highest remaining concentration (450 µg/mL) and an intermediate concentration of (300 µg/mL) in the presence of S-9 were not selected to determine viability and 6TG resistance (there was excessive toxicity at 450 µg/mL and one culture at 300 µg/mL had a zero count on survival plates which may have been attributable to toxicity or a dilution error prior to plating). All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 450 µg/mL in the absence of S-9 and 400 µg/mL in the presence of S-9 which gave 13% and 12% RS, respectively (see table).

Genotoxicity (see tables)
The acceptance criteria were met and the study was accepted as valid.
When tested up to toxic and/or precipitating concentrations, no statistically significant increases in MF, compared to the vehicle control MF values, were observed at any N,N’-Dibutylthiourea concentration analysed in the absence and presence of S-9. A statistically significant linear trend (p=0.01) was observed in the presence of S-9 but in the absence of any statistically significant increases in MF at any concentration analysed, this observation was considered not biologically relevant.

Applicant's summary and conclusion

Conclusions:

It is concluded that N,N’-Dibutylthiourea did not induce mutation at the hprt locus in mouse lymphoma L5178Y cells when tested up to toxic and/or precipitating concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

Executive summary:

N,N’-Dibutylthiourea was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO). A 3 hour treatment incubation period was used for each experiment.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 37.50 to 1200 µg/mL (a precipitating treatment concentration). The highest concentrations to give >10% relative survival (RS) were 600 µg/mL in the absence of S-9 and 300 µg/mL in the presence of S-9 (limited by the appearance of post treatment precipitate in the absence of S-9), which gave 11% and 39% RS, respectively.

In the Mutation Experiment eleven concentrations, ranging from 50 to 700 µg/mL in the absence of S-9 and from 25 to 600 µg/mL in the presence of S-9, were tested.

Seven days after treatment the highest concentrations selected to determine viability and 6TG resistance were 450 µg/mL in the absence of S-9 and 400 µg/mL in the presence of S-9 (limited by the appearance of post treatment precipitate in the absence of S-9), which gave 13% and 12% RS, respectively. Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures

fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

When tested up to toxic and/or precipitating concentrations, no statistically significant increases in MF, compared to the vehicle control MF values, were observed at any N,N’-Dibutylthiourea concentration analysed in the absence and presence of S-9. A statistically significant linear trend (p=0.01) was observed in the presence of S-9 but in the absence of any statistically significant increases in MF at any concentration analysed, this observation was considered not biologically relevant.

It is concluded that N,N’-Dibutylthiourea did not induce mutation at the hprt locus in mouse lymphoma L5178Y cells when tested up to toxic and/or precipitating concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.