Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The three in vitro recommended tests were performed on the registered substance.
The Ames test, HPRT test and in vitro micronucleus test showed negative results in presence and in absence of metabolic activation.

Link to relevant study records

Referenceopen allclose all

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
Reference:
Composition 0
Qualifier:
according to
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 information:
Composition 1
Target gene:
hprt locus
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (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 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:
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.
Negative controls:
no
Solvent 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 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.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
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.
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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July - August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
not applicable
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell lines (if applicable):
n/a
Additional strain characteristics:
not applicable
Cytokinesis block (if used):
n/a
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:
-Mutation experiment 1: 5, 16, 50, 160, 500, 1600 and 5000 µg/plate
-Mutation experiment 2: The maximum test concentration was reduced to 3200 µg/plate based on toxicity observed in Mutation Experiment 1. A narrowed concentration interval was also employed covering the range of 50-3200 µg/plate.
Vehicle:
dimethyl sulphoxide (DMSO)
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene : with S9 (TA100, TA102, TA1535, TA1537)
Details on test system and conditions:
N,N’-Dibutylthiourea was tested for mutation (and toxicity) in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), in two separate experiments, at the concentrations detailed previously, using triplicate plates without and with S-9 for test article, vehicle and positive controls. These platings were achieved by the following sequence of additions to molten agar at 45±1°C:
• 0.1 mL bacterial culture
• 0.1 mL of test article solution/vehicle control or 0.05 mL of positive control
• 0.5 mL 10% S-9 mix or buffer solution
followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1°C protected from light for 2 or 3 days.
Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.
As the results of Mutation Experiment 1 were negative, treatments in the presence of S-9 in Mutation Experiment 2 included a pre-incubation step. Quantities of test article, vehicle control solution (reduced to 0.05 mL) or positive control, bacteria and S-9 mix detailed above, were mixed together and incubated for 20 minutes at 37±1°C, with shaking, before the addition of 2 mL molten agar at 45±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected
in the assay.
Volume additions for the Mutation Experiment 2 pre-incubation treatments were reduced to 0.05 mL due to the vehicle (DMSO) employed in this study. This, and some other organic vehicles, are known to be near to toxic levels when added at volumes of 0.1 mL in this assay system when employing the pre-incubation methodology. By reducing the addition volume to 0.05 mL per plate, it was hoped to minimise or eliminate any toxic effects of the vehicle that may have otherwise occurred.

Toxicity assessment : The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.
Rationale for test conditions:
The assay was to be considered valid if all the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges
2. The positive control chemicals induced increases in revertant numbers of =1.5-fold (in strain TA102), =2-fold (in strains TA98 and TA100) or =3-fold (in strains TA1535 and TA1537) the concurrent vehicle control confirming discrimination between different strains, and an active S-9 preparation
Evaluation criteria:
For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was =1.5-fold (in strain TA102), =2-fold (in strains TA98 or TA100) or =3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values
2. The positive trend/effects described above were reproducible.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met.
Statistics:
No
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
Toxicity, Solubility and Concentration Selection :
Mutation Experiment 1 : evidence of toxicity in the form of a complete killing of the test bacteria was observed at 5000 µg/plate in all strains in the absence and presence of S-9. A slight thinning of the background bacterial lawn was also noted at 1600 µg/plate in strain TA98 in the absence of S-9, in strain TA100 in the presence of S-9 and in strains TA1535, TA1537 and TA102 in both the absence and presence of S-9.
Mutation Experiment 2 : evidence of toxicity in the form of slight thinning of the background bacterial lawn, very thin background bacterial lawn or a complete killing of the test bacteria was observed at 3200 µg/plate in all strains in the absence and presence of S-9. Slight thinning of the background bacterial lawn was observed at 1600 µg/plate in strain TA100 in the absence of S-9 and in strains TA1535, TA1537 and TA102 in the absence and presence of S-9. Slight thinning of the background bacterial lawn was also observed in strain TA102 at 800 µg/plate in the absence of S-9 and at 400 and 800 µg/plate in the presence of S-9.

Data Acceptability and Validity :
From the data it can be seen that vehicle control counts fell within the laboratory’s historical ranges with the exception of a single replicate count in strain TA1537 in the presence of S-9 in mutation Experiment 1, which fell below the historical control range. As this was only slightly below the range and the mean and remaining counts fell within range, the data were considered characteristic of the strain and were accepted as valid.
The positive control chemicals all induced increases in revertant numbers of =1.5-fold (in strain TA102), =2-fold (in strains TA98 and TA100) or =3-fold (in strains TA1535 and TA1537) the concurrent vehicle controls confirming discrimination between different strains, and an active S-9 preparation. The study therefore demonstrated correct strain and assay functioning and was accepted as valid.

Mutagenicity results :
Following N,N’-Dibutylthiourea treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were =1.5-fold (in strain TA102), =2-fold (in strains TA98 and TA100) or =3-fold (in strains TA1535 and TA1537) the concurrent vehicle control.
Conclusions:
It was concluded that N,N’-Dibutylthiourea did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These
conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).
Executive summary:

N,N’-Dibutylthiourea was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments. All N,N’-Dibutylthiourea treatments in this study were performed using formulations prepared in anhydrous analytical grade dimethyl sulphoxide (DMSO).

Mutation Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of N,N’-Dibutylthiourea at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity was observed at 1600 and/or 5000 µg/plate in all strains in the absence / presence of S-9.

Mutation Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration was reduced to 3200 µg/plate based on toxicity observed in Mutation Experiment 1. A narrowed concentration interval was also employed covering the range of 50-3200 µg/plate, in order to examine more closely those concentrations of N,N’-Dibutylthiourea approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system.

Following these treatments, evidence of toxicity was observed at 1600 and/or 3200 µg/plate in all strains in the absence / presence of S-9 and also at 400 and/or 800 µg/plate in strain TA102 in the absence / presence of S-9.

No precipitation was observed on the test plates following incubation. Vehicle and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies fell within acceptable ranges

for vehicle control treatments, and were elevated by positive control treatments. Following N,N’-Dibutylthiourea treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were =1.5-fold (in strain TA102), =2-fold (in strains TA98 or TA100) or =3-fold (in strains TA1535 or TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any N,N’-Dibutylthiourea mutagenic activity in this assay system.

It was concluded that N,N’-Dibutylthiourea did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These

conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June-October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Test material information:
Composition 1
Target gene:
n/a
Species / strain:
lymphocytes: human
Details on mammalian cell lines (if applicable):
Blood from two healthy, non-smoking female volunteers from a panel of donors at Covance was used for each experiment.
No donor was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All donors are non-smokers and are not heavy drinkers of alcohol. Donors were not taking any form of medication (contraceptive pill excluded). The measured cell cycle time of the donors used at Covance, Harrogate falls within the range 13±2 hours. For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes on the day prior to culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.
Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 8.5 mL pre-warmed (in an incubator set to 37±1°C) HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin / streptomycin, so that the final volume following addition of S-9 mix/KCl and the test article in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37±1°C for approximately 48 hours and rocked continuously.
Metabolic activation:
with and without
Metabolic activation system:
liver of male Sprague Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
Range finder : 7.256 - 2000 µg/mL
Main study / 3h of treatment without S9 : 10 - 700 µg/ml
Main study / 3h of treatment with S9 : 10 - 700 µg/ml
Main study / 24h of treatment without S9 : 10 - 125 µg/ml

See below for the justification of the top dose used in the main experiment.
Vehicle:
Preliminary solubility data indicated that N,N' Dibutylthiourea was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to 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. A maximum concentration of 2000 µg/mL was selected for the cytotoxicity Range-Finder Experiment, in order that treatments were performed up to the maximum recommended concentration according to current regulatory guidelines (OECD, 2016). Concentrations for the Micronucleus Experiment were selected based on the results of this cytotoxicity Range-Finder Experiment.
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: vinlastine (treatment of 24h without S9)
Details on test system and conditions:
S-9 mix or KCl (1 mL per culture) was added appropriately. Cultures were treated with the test article, vehicle or positive controls (0.1 mL per culture).
The final culture volume was 10 mL. Cultures were incubated at 37±1°C for the designated exposure time.

Number of cultures (main study) :
vehicule control : 4 by condition
test article : 2 by condition
positive control : 2 by condition

For removal of the test article, cells were pelleted (approximately 300 g, 10 minutes), washed twice with sterile saline (pre-warmed in an incubator set to 37±1°C), and resuspended in fresh pre-warmed medium containing foetal calf serum and penicillin / streptomycin. Cyto-B (formulated in DMSO) was added to post wash-off culture medium to give a final concentration of 6 µg/mL per culture.

Hours after culture initiation :
-Addition of test article : 48h
-Removal of test article : 51h (3h treatment) or 72h (24h of treatment)
-Addition of Cyto-B: 52h (3h treatment) or 73 (24h of treatment)
-harvest time: 72h (3h treatment) or 96h (24h of treatment)

Selection of concentrations for micronucleus analysis :
Slides were examined, uncoded, for RI to a minimum of 500 cells per culture to determine whether chemically induced cell cycle delay or toxicity had occurred.
The highest concentration selected for micronucleus analysis following all treatment conditions was one at which 50-60% cytotoxicity was achieved (OECD, 2016). Analysis of slides from highly toxic concentrations was avoided.
Slides from the highest selected concentration and two or three lower concentrations were taken for microscopic analysis, such that a range of cytotoxicity from maximum to little was covered.
The positive control concentrations analysed did not exceed the cytotoxicity limits for the test article concentration selection.

Scoring was carried out using fluorescence microscopy.
After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei (MNBN cells) in each culture were obtained.
Rationale for test conditions:
Acceptance criteria :
The assay was to be considered valid if the following criteria were met:
1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures, particularly where no positive responses were seen
2. The frequency of MNBN cells in vehicle controls fell within the current 95th percentile of the observed historical vehicle control (normal) ranges
3. The positive control chemicals induced statistically significant increases in the proportion of cells with micronuclei. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the normal range
4. A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at the time of harvest
5. The maximum concentration analysed under each treatment condition met the criteria specified in the study protocol
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed (positive trend test).
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al., 1990). Biological relevance was taken into account, for example consistency of response within and between concentrations, or effects occurring only at very toxic concentrations (Thybaud et al., 2007).
Statistics:
yes
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
No marked changes in osmolality (shifts of greater than 50 mOsm/kg) or pH (shifts of greater than 1 pH unit) were observed in post treatment medium samples at the highest three concentrations tested (720, 1200 and 2000 µg/mL) as compared to the concurrent vehicle controls (individual data not reported).
The results of the cytotoxicity Range-Finder Experiment were used to select suitable maximum concentrations for the Micronucleus Experiment.

Validity of the study :
1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures for the majority of treatments. The one exception to this was following 3+21 hour +S-9 treatment where weak but statistically significant heterogeneity was observed. However, this was predominantly attributable to a single vehicle control culture (Replicate 'A') that exhibited an elevated MNBN cell response. As all remaining vehicle and all test article treated cultures (all concentrations) exhibited MNBN cell values within normal ranges, the interpretation of the data was clear. As such, the observed heterogeneity did not impact on the validity of the study.
2. The frequency of MNBN cells in vehicle controls fell within the normal range for the majority of treatments The single exception to this was observed following 3+21 hour +S-9 treatment where the replicate 'A' culture exhibited 1.1% MNBN cells, marginally exceeding the normal range (0.2 to 1.07% MNBN cells). However, as this increase was marginal and was not observed in the 'B', 'C' and 'D' cultures with an overall vehicle mean MNBN cell value (0.53% MNBN cells) that fell within the normal range, the vehicle data was considered acceptable
3. The positive control chemicals induced statistically significant increases in the proportion of MNBN cells. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the normal range
4. A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at the time of harvest
5. The maximum concentration analysed under each treatment condition met the criteria specified in study protocol.

Analysis of data :
Treatment of cells with N,N' Dibutylthiourea in the absence and presence of S 9 resulted in frequencies of MNBN cells which were similar to and not significantly (p=0.05) higher than those observed in concurrent vehicle controls for the majority of all concentrations analysed (all treatments). A single exception to this was observed at the highest concentration analysed (200 µg/ml, inducing 51% cytotoxicity) following 3+21 hour -S-9 treatment. However, although statistically significant, this increase was small (0.55% MNBN cells) and set against a low concurrent vehicle control response (0.25% MNBN cells) and did not exceed normal ranges (0.2 to 1.0 MNBN cells). The MNBN cell frequency of both replicate cultures for all N,N' Dibutylthiourea treated cultures (all concentrations, all treatments) fell within normal ranges. As such, this isolated small statistically significant increase was not considered of biological importance.
Conclusions:
It is concluded that N,N'-Dibutylthiourea did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of an Aroclor-induced rat liver metabolic activation system (S-9). Maximum concentrations analysed induced between 51-54% cytotoxicity (consistent with current regulatory guidelines for the in vitro micronucleus assay).
Executive summary:

N,N'-Dibutylthioureawas tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of twofemaledonors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest concentrations tested in the Micronucleus Experiment were determined following a preliminary cytotoxicity Range-Finder Experiment.

Treatment of cells with N,N'-Dibutylthioureain the absence and presence of S-9 resulted in frequencies of MNBN cells which were similar to and not significantly (p=0.05) higher than those observed in concurrent vehicle controls for the majority of all concentrations analysed (all treatments). A single exception to this was observed at the highest concentration analysed (200 µg/ml, inducing 51% cytotoxicity) following 3+21 hour -S-9 treatment. However, although statistically significant, this increase was small (0.55% MNBN cells) and set against a low concurrent vehicle control response (0.25% MNBN cells) and did not exceed the normal range (0.2 to 1.0 MNBN cells). The MNBN cell frequency of both replicate cultures for all N,N'-Dibutylthioureatreated cultures (all concentrations, all treatments) fell within normal ranges. As such, this isolated small statistically significant increase was not considered of biological importance.

It is concluded that N,N'-Dibutylthiourea did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of an Aroclor-induced rat liver metabolic activation system (S-9). Maximum concentrations analysed induced between 51-54% cytotoxicity (consistent with current regulatory guidelines for the in vitro micronucleus assay).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

in vitro gene mutation study in bacteria / OECD 471 (Dreher 2017) :

N,N’-Dibutylthiourea was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments. All N,N’-Dibutylthiourea treatments in this study were performed using formulations prepared in anhydrous analytical grade dimethyl sulphoxide (DMSO).

Mutation Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of N,N’-Dibutylthiourea at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity was observed at 1600 and/or 5000 µg/plate in all strains in the absence / presence of S-9.

Mutation Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration was reduced to 3200 µg/plate based on toxicity observed in Mutation Experiment 1. A narrowed concentration interval was also employed covering the range of 50-3200 µg/plate, in order to examine more closely those concentrations of N,N’-Dibutylthiourea approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, evidence of toxicity was observed at 1600 and/or 3200 µg/plate in all strains in the absence / presence of S-9 and also at 400 and/or 800 µg/plate in strain TA102 in the absence / presence of S-9.

Following N,N’-Dibutylthiourea treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed.

It was concluded that N,N’-Dibutylthiourea did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study.

in vitro gene mutation study in mammalian cells / OECD 476 (Lloyd 2017) :

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 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.

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.

in vitro micronucleus study in mammalian cells / OECD 487 (Whitwell 2017) :

N,N'-Dibutylthioureawas tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of twofemaledonors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest concentrations tested in the Micronucleus Experiment were determined following a preliminary cytotoxicity Range-Finder Experiment.

Treatment of cells withN,N'-Dibutylthioureain the absence and presence of S-9 resulted in frequencies of MNBN cells which were similar to and not significantly (p=0.05) higher than those observed in concurrent vehicle controls for the majority of all concentrations analysed (all treatments). A single exception to this was observed at the highest concentration analysed (200 µg/ml, inducing 51% cytotoxicity) following 3+21 hour -S-9 treatment. However, although statistically significant, this increase was small (0.55% MNBN cells) and set against a low concurrent vehicle control response (0.25% MNBN cells) and did not exceed the normal range (0.2 to 1.0 MNBN cells). The MNBN cell frequency of both replicate cultures for allN,N'-Dibutylthioureatreated cultures (all concentrations, all treatments) fell within normal ranges. As such, this isolated small statistically significant increase was not considered of biological importance.

It is concluded that N,N'-Dibutylthiourea did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of an Aroclor-induced rat liver metabolic activation system (S-9). Maximum concentrations analysed induced between 51-54% cytotoxicity (consistent with current regulatory guidelines for thein vitromicronucleus assay).

Additional information

Justification for classification or non-classification

Based on the available data, no classification for mutagenicity is required for the registered substance according to the Regulation EC N°1272/2008.