Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

Bacterial reverse mutation test : Sarlang (2012)

The objective of this study (OECD 471) was to evaluate the potential of the test item to induce reverse mutation inSalmonella typhimurium.

The first experiment (with and without S9 mix) and the second experiment without S9 mix were performed according to the direct plate incorporation method, contrary to the second, third and fourth experiments with S9 mix, which were performed according to the pre-incubation method (60 minutes, 37°C). Five strains of bacteriaSalmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to six dose-levels of the test item (three plates/dose‑level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item was dissolved in dimethylsulfoxide (DMSO).

The number of revertants for the vehicle and positive controls met the acceptance criteria. The study was therefore considered to be valid.

Since the test item was found poorly soluble in the preliminary test, the choice of the highest dose-level was based on the level of precipitate, according to the criteria specified in the international guidelines.

Experiments without S9 mix: The selected treatment-levels were 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for the first and second experiments. A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 1250 µg/plate.

In the first experiment, a moderate toxicity (thinning of the bacterial lawn) was noted at dose‑levels of 1250 µg/plate in the strains TA 1535 and TA 1537, and at 2500 µg/plate in the TA 100 strain.

In the second experiment, a moderate toxicity (thinning of the bacterial lawn) was noted at 2500 µg/plate in the TA 98 strain.

No noteworthy toxicity was noted towards the strain TA 102 in either experiment.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.

Experiments with S9 mix : In the first experiment performed using the direct plate incorporation method, a moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 1250 µg/plate.

Using the pre-incubation method, a moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >=1250 µg/plate, except for the strains TA 100 and TA 1537 in the second experiment where a moderate precipitate was observed at 1250 µg/plate only. In the fourth experiment, a strong toxicity (decrease in the number of revertants) was noted at 2500 µg/plate towards the strain TA 1537.

No noteworthy toxicity was noted towards the other strains used.

 Noteworthy increases in the number of revertants were noted at 2500 µg/plate in the strains TA 1535, TA 1537 and TA 100 in the second experiment performed with S9 mix. These increases exceeded the threshold of 2-fold the vehicle control (2.6-fold the vehicle control for the strain TA 100) and of 3-fold the vehicle control (up to 30.3-fold the vehicle control for the strains TA 1535 and TA 1537). Two additional experiments were performed using the same experimental conditions (pre-incubation method) and a closer range of dose-levels to check the reproducibility and therefore the reliability of these increases. In these experiments, no increases in the number of revertants were noted at any of the dose-levels tested. Thus, the increases noted in the second experiment were not reproducible in two additional independent experiments. Consequently, they were not considered as biologically relevant.

The test item did not show any mutagenic activity in the bacterial reverse mutation test withSalmonella typhimurium, in the presence or in the absence of a rat metabolizing system.

 

In vitro in mammalian cells gene mutation test (OECD 476) : Sarlang (2012)

The objective of this study (OECD 476) was to evaluate the potential of the test item to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/-mouse lymphoma cells.

After a preliminary toxicity test, 1,3-Diphenyl-2-thiourea, was tested in two independent experiments, with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2).The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

The test item was dissolved in dimethylsulfoxide (DMSO).

With a few exceptions which were not considered to have a biological impact on the validity of the study, the Cloning Efficiencies (CE2), the Suspension Growths (SG) and the mutation frequencies of the vehicle and positive controls were considered as specified in the acceptance criteria. The study was therefore considered to be valid.

Since the test item was toxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in Adj. RTG).

At the end of the 3-hour treatment (first experiment without S9), a precipitate was noted at dose-level of 200 µg/mL.

Following the 3-hour treatment (withour S9), a slight to severe toxicity was induced at dose-levels comprise between 25 and 100 µg/mL, as shown by a 34-98% decrease in the Adj. RTG. At 200 µg/mL, no evaluation of the decrease in the Adj. RTG could be performed since no cell was viable after the expression period.Following the 24-hour treatment (second experiment without S9), a slight to severe toxicity was induced at dose‑levels ≥ 12.5 µg/mL, as shown by a 30-100% decrease in the Adj. RTG.

Following the 3-hour treatment without S9, a slight increase in the mutation frequency was observed at dose‑levels ≥ 50 µg/mL. This increase did not exceed the GEF even at the too cytotoxic dose-level of 100 µg/mL (inducing a 98% decrease in the Adj. RTG). Thus this result did not meet the criteria of a positive response.Following the 24-hour treatment without S9, no increase in the mutation frequency was observed up to (and even above) the dose-level of 25 µg/mL, inducing a 90% decrease in the Adj. RTG. Thus this result (without S9) did not meet the criteria of a positive response.

In the first and second experiments with S9, a marked to severe toxicity was induced at dose-levels ≥ 12.5 µg/mL, as shown by a 68-100% decrease in the Adj. RTG.At the end of the first experiment, no precipitate was noted at any dose-levels.

In the first experiment with S9, dose-related increases in the mutation frequency were observed at dose‑levels ≥ 6.25 µg/mL. These increases exceeded the global evaluation factor of +126 x 10^-6 at the dose-level of 12.5 µg/mL, inducing an acceptable level of cytotoxicity (68% decrease in the Adj. RTG).Thus this result met the criteria of a positive response.

In the second experiment with S9, dose-related increases in the mutation frequency were observed at dose‑levels ≥ 6.25 µg/mL. These increases exceeded the GEF at the dose-levels of 12.5 and 18.8 µg/mL, inducing acceptable levels of cytotoxicity (76 and 90% decrease in the Adj. RTG, respectively). Thus this result met the criteria of a positive response.

Compared to the vehicle control, the mutation frequencies in the second experiment with S9 were increased of up to 82 x 10^-6 and 130 x 10^-6, for the large and small colonies respectively at 12.5 µg/mL, and 128 x 10^-6 and 220 x 10^-6, for the large and small colonies respectively at 18.8 µg/mL. This might indicate that the test item induced chromosome damages as well as point mutations.

To conclude the test item showed a mutagenic activity in the mouse lymphoma assay, in the presence of metabolizing system, whereas it did not in the absence of metabolic activation. Moreover, a high number of small colonies were observed in the positive second experiment, this might indicate that the test item induced chromosome damages as well as point mutations.

 

In vivo micronucleus test : Sire (2012)

The objective of this study (OECD 474) was to evaluate the potential of the test item to induce damage to the chromosomes or the mitotic apparatus in rat bone marrow cells.

In the main study, three groups of five male and five female Sprague-Dawley rats received two oral treatments of 1,3-Diphenyl-2-thiourea at dose-levels of 500, 1000 and 2000 mg/kg/day, at a 24-hour interval. For the high-dose group only, two supplementary males and three supplementary females were also treated with the test item in case of mortality.One group of five males and five females received the vehicle (corn oil) under the same experimental conditions, and acted as control group.

One group of five males and five females received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 15 mg/kg/day.For each animal, the number of the Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 Polychromatic Erythrocytes. The Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio was established by scoring a total of 1000 Erythrocytes (PE + NE).

According to the criteria specified in the international guidelines, since no toxic effects were observed at 2000 mg/kg/day in the preliminary test, this dose-level was selected as the top dose‑level for the main test. The two other selected dose-levels were 500 and 1000 mg/kg/day.

No mortalities and no clinical signs were observed at any of the tested dose-levels during the study.

The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with our historical data.

Cyclophosphamide induced a significant increase (p < 0.001 males and p < 0.05 females) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered to be valid.

The test item did not induce any noteworthy decrease in the PE/NE ratios when compared to the vehicle control group.The mean values of MPE in the test item-treated groups were found equivalent to those of the vehicle group. These results met the criteria of a negative response.

The test item did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells after two oral administrations, 24-hour apart, at the dose-levels of 500, 1000 and 2000 mg/kg/day.

 


Justification for selection of genetic toxicity endpoint
Endpoint conclusion is based on the evaluation of all the genotoxicity studies.

Short description of key information:
Several studies are available to evaluate the mutagenicity of DPTU. Ames test showed negative results with and without S9. The mouse lymphoma assay (OECD 476) showed positive response with metabolic activation, with a majority of small colonies which suggested a possible clastogenic effect. That's why an in vivo micronucleus test was performed by oral route in rat: no clastogenic effect was observed in this in vivo study. Therefore DPTU is not considered to be mutagenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of all genotoxicity studies, DPTU is not considered to be mutagenic, therefore no classification is required according to the Regulation EC 1272/2008 or the Directive 67/548/EEC.