Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro genetic data is available for Toluene-4-sulphonohydrazide (TSH):


 



  • Toluene-4-sulphonohydrazide (TSH) was found to be genotoxic in bacteria cells in two reverse mutation assays (OECD 471) with Salmonella typhimurium and Escherichia coli with and without metabolic activation (S9 mix).


 



  • Toluene-4-sulphonohydrazide (TSH) was found to be genotoxic in mammalian cells in the mouse lymphoma mutagenicity assay (OECD 476) without metabolic activation (S9 mix).


 



  • Toluene-4-sulphonohydrazide (TSH) was found not to induce chromosomal aberrations in the in vitro mammalian chromosomal aberration test (OECD 473) using Chinese hamster ovary (CHO) with and without metabolic activation (S9 mix).


 


In summary, Toluene-4-sulphonohydrazide (TSH) was found to be mutagenic in two bacteria cell assays (OECD 471) with and without metabolic activation (S9 mix) as well as in mammalian cells (OECD 476) without metabolic activation (S9 mix).  Toluene-4-sulphonohydrazide (TSH) did not induce chromosomal aberrations using Chinese hamster ovary (CHO) in accordance to OECD 473 with and without metabolic activation (S9 mix).


 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
not specified
Type of assay:
other: Mouse lymphoma mutagenicity assay
Target gene:
NA
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, male rat liver, Aroclor 1254 induced
Test concentrations with justification for top dose:
100-2400 µg/mL
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Evaluation criteria:
Mutant frequencies were expressed as mutants per 106 surviving cells. Although there are several different methods for evaluating mouse lymphoma data, results from this study were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
not specified
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
At 1000 ug/mL
Cytotoxicity / choice of top concentrations:
not specified
Conclusions:
p-toluenesulfonic hydrazide (TSH) showed mutagenic effects in mouse lymphoma L5178Y at 1000 µg/mL without S9 metabolic activation system. The results were equivocal with S9 metabolic activation system
Executive summary:

The genetic toxicity of p-toluenesulfonic hydrazide (TSH)was evaluation in a Mouse lymphoma mutagenicity assay. The study was performed in equivalence to OECD guideline 476.


TSH showed mutagenic effects in mouse lymphoma L5178Y at 1000 µg/mL without S9 metabolic activation system. The results were equivocal with S9 metabolic activation system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Arochlor 1254-induced rat liver homogenate (S9)
Test concentrations with justification for top dose:
Trial 1 (10 h): 0, 0.1, 0.3, 0.6, 1.0 mM
Trial 2 (16 h): 0, 0.1, 0.3, 0.6, 1.0 mM

The top dose was the highest dose, at which an adequate number of metaphase cells could be recovered
Vehicle / solvent:
serum free complete medium
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid

In two seperate trials, there were no significant increase in the percentage of cells with chromosomal aberrations or the number of chromosomal aberrations per cell measured either 10 or 16 h following treatment. In all experiments, the top dose was the highest dose at which an adequate number of metaphase cells could be recovered

Conclusions:
TSH does not induce chromosomal aberrations in CHO cells.
Executive summary:

In this study the mutagenic acitivity of TSH was determined in the in vitro chromosomal aberration assay using Chinese hamster ovary (CHO) cells. TSH did not induce chromosomal aberrations in the CHO cells. In two seperate trials, there were no significant increase in the percentage of cells with chromosomal aberrations or the number of chromosomal aberrations per cell measured either 10 or 16 h following treatment. In all experiments, the top dose was the highest dose at which an adequate number of metaphase cells could be recovered. The results demonstrate that TSH does not induce chromosomal aberrations in CHO cells.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: TA 97, TA 98; TA 100, TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Trial 1: control (DSMO), 5, 10, 50, 100, 500, 1000
Trial 2: control (Acetone), 7.5, 25, 75, 250, 750, 2500
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:
Trial 1: DMSO
Trial 2: acetone
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Key result
Species / strain:
other: S. typhimurium TA 97, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
In this study the mutagenic activity of TSH was determined in the salmonella/mammalian microsome assay. TSH induced gene mutations both with and without metabolic activation. This study indicates that TSH is mutagenic in vitro.
Executive summary:

In this study the mutagenic acitivity of TSH was determined in the salmonella/mammalian microsome assay. TSH induced gene mutations both with and without metabolic activation. This study indicates that TSH is mutagenic in vitro.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
bacterial forward mutation assay
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
NA
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Test concentrations with justification for top dose:
100-10000 µg/plate
Untreated negative controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
NA
Evaluation criteria:
To be considered positive, it had to induce at least a doubling in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose
response to increasing concentrations of the test chemical.
Statistics:
No data
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
at 100 ug/plate
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
p-toluenesulfonic hydrazide (TSH) showed mutagenic effects in Salmonella typhimurium TA 100 at 100 µg/plate with and without metabolic activation system.
Executive summary:

The genetic toxicity of p-toluenesulfonic hydrazide (TSH) was evaluation in a bacterial reverse mutation assay. The study was performed in equivalence to OECD guideline 471.


TSH showed mutagenic effects in Salmonella typhimurium TA 100 at 100  µg/plate with and without metabolic activation system.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo genetic data is available for Toluene-4-sulphonohydrazide (TSH):


 


- Toluene-4-sulphonohydrazide (TSH) was tested for genetic toxicity in vivo in accordance to the most recent OECD 489 guideline (Comet Assay) using samples from Liver, Duodenum and Glandular Stomach. Based on the results of a dose-range finding test concentrations of 250 mg/kg/day for male animals was selected as maximum dose for the main test (maximum tolerated dose). Dose levels used were Group 2 (62.5 mg/kg), Group 3 (125 mg/kg) and Group 4 (250 mg/kg).  


 


No statistically significant increase in the mean Tail Intensity (%) was observed in Liver, Duodenum and Glandular Stomach cells of test item treated male treated animals compared to the vehicle treated animals. A statistically significant increase was observed in Duodenum cells of the animals treated with 62.5 mg/kg compared to the vehicle control treated animals. However, no significant concentration-related increase was observed, and the results are within the 95% control limits of the negative historical control data range.  Therefore, the increase was considered not of biological relevance.


 


In conclusion, Toluene-4-sulphonohydrazide (TSH) did not show any evidence of causing an increase in DNA strand breaks in Liver, Duodenum and Glandular Stomach cells when sampled approximately 3-4 hours post dosing, of male rats, dosed via oral gavage for two consecutive days up to a dose of 250 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines). Thus, Toluene-4-sulphonohydrazide (TSH) was found not to be genotoxic (In vivo) in the Comet assay (OECD 489). 


 


 

Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): The Sponsor has appropriate documentation on file concerning the method of synthesis, fabrication or derivation of the test item, and this information is available to the appropriate regulatory agencies should it be requested.
- lot/batch number of test material: 202102001
- Purity, including information on contaminants, isomers, etc.: 99.5%
- Physical description: White powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability analyses performed previously demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for (Test Facility Study No. 20285169).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final concentration of a dissolved solid, stock liquid or gel: No correction was made for the purity/composition of the test item. The test item was dissolved (clear colorless) in propylene glycol (Merck, Darmstadt, Germany). The specific gravity of propylene glycol is 1.036 g/mL. Test item concentrations were treated with ultra-sonic waves to obtain a clear solution. Test item concentrations were dosed within 4 hours after preparation.

NEGATIVE CONTROL: The negative control was propylene glycol, the vehicle of the test item.
The route and frequency of administration and the volume administered of the negative control were the same as those of the test item.

POSITIVE CONTROL: The positive control was ethyl methanesulfonate (EMS, Sigma Aldrich, Steinheim, Germany) at 200 mg/kg body weight dissolved in physiological saline. EMS was used within 2 hours after preparation.

SAMPLE COLLECTION AND ANALYSIS:
Dose formulation samples were collected for analysis using a validated analytical procedure.
Acceptance Criteria of the concentration and homogeneity analysis:
For concentration: mean sample concentration results within or equal to ± 10% (solutions) of theoretical concentration.
For homogeneity, relative standard deviation (RSD) of concentrations of  10% for each group.
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI (Han)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 6-7 weeks
- Number of males: 3(dose range finding), 26 (main study - 5 males animals were used in each treatment group, except for the positive control (3 animals). In the highest dose group 3 additional animals were included to replace in case of unexpected deaths)
- Number of females: 3 (dose range finding)
- Weight at study initiation: The body weights of the rats at the start of the treatment were within 20% of the sex mean. The mean body weights were for males 142 ± 7.0 g and the range for males 129 – 151 g.
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: Polycarbonate cages (Makrolon MIV type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Up to 5 animals of the same sex and same dosing group were housed together.
- Diet (e.g. ad libitum): Ad libitum, except during designated procedures.
- Water (e.g. ad libitum): Ad libitum.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.9 to 21.5°C
- Humidity (%): 51 to 73%.
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES:
From:
Study Initiation Date: 18. June 2021
Initiation of Dosing: 21. June 2021
To: Completion of In-life: 29. July 2021

The Wistar-Han rat was chosen as the animal model for this study as it is an accepted rodent species for nonclinical toxicity test by regulatory agencies.
The total number of animals to be used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study was designed such that it does not require an unnecessary number of animals to accomplish its objectives.
The study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 1 of project license AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Propylene glycol (Merck, Darmstadt, Germany).
- Justification for choice of solvent/vehicle: Standardly used in comet assay.
- Amount of vehicle (if gavage or dermal): No information
- Lot/batch no. (if required): No information
- Purity: No information
Details on exposure:


TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Constant volume or concentration used: yes/no
- For solids, paste formed: yes/no

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes/no
Duration of treatment / exposure:
2 consecutive days
Frequency of treatment:
Daily - a total of two doses were given
Post exposure period:
Approximately 3-4 hours after the last dose the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia tissues were isolated.
Dose / conc.:
250 mg/kg bw/day
Remarks:
The highest dose was 250 mg/kg body weight, i.e. the Maximum Tolerated Dose (MTD; described as the dose that will not kill the animals but will provoke signs of toxicity).
Dose / conc.:
125 mg/kg bw/day
Dose / conc.:
62.5 mg/kg bw/day
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
No. of animals per sex per dose:
Based on the results of the dose-range finding test a study with one sex was performed. Since there were no substantial differences in toxicity between sexes, only male animals were used in the main study.
Five (5) animals in each dose group, except for the positive control (3 animals). In the highest dose group 3 additional animals were included to replace in case of unexpected deaths.
Control animals:
yes, concurrent vehicle
Positive control(s):
A positive control group was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight.
Tissues and cell types examined:
Approximately 3-4 hours after the last treatment with the test item, vehicle or EMS Liver, Duodenum and Glandular Stomach were collected/isolated and examined for DNA damage with the alkaline Comet assay.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose-range finding test.

DETAILS OF SLIDE PREPARATION: To the cell suspension, melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added (ratio 10:140). The cells were mixed with the LMAgarose and 50 µL was layered on a pre-coated Comet slide (Trevigen) in duplicate. Three slides per tissue per animal were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 15-23 minutes in the refrigerator in the dark until a clear ring appears at the edge of the Comet slide area.

METHOD OF ANALYSIS (lysis, electrophoresis and staining of the slides):
The cells on the slides were overnight (approximately 16-18 h) immersed in pre-chilled lysis solution in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution for 20-30 minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 0.7 Volt/cm. The electrophoresis was performed for 20 or 30 minutes under constant cooling (actual temperature 4.0°C). After electrophoresis the slides were immersed/rinsed in neutralization buffer for 30 minutes. The slides were subsequently immersed for 7-9 minutes in Absolut ethanol (99.6%, Merck) and allowed to dry at room temperature. The slides were stained for approximately 5 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark and fixed with a coverslip.

No histotechnology and histopathology was needed.
Evaluation criteria:
To prevent bias, slides were randomly coded (per tissue) before examination of the Comets. An adhesive label with study identification number and code were placed over the marked slide. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system (Perceptive instruments Ltd, Suffolk, United Kingdom). One hundred fifty Comets (50 comets of each replicate LMAgarose circle) were examined per sample.

The following criteria for scoring of Comets were used:
• Only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
• Cells that showed overlap or were not sharp were not scored.

In addition, the frequency of hedgehogs was determined and documented based on the visual scoring of at least 150 cells per tissue per animal. The occurrence of hedgehogs was scored in all treatment groups and the control. Since there was no effect of the test item Hedgehogs data was not reported and maintained in the raw data.



ACCEPTABILITY CRITERIA:
The in vivo comet is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The positive control EMS should produce at least a statistically significant increase in the percentage Tail Intensity compared to the vehicle treated animals. The response should be compatible with the data in the historical control database.
c) Adequate numbers of cells and doses have been analysed
d) The highest test dose is the MTD
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the comet assay data.
A test item is considered positive in the Comet assay if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative in the Comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.
As the Dunnett’s t-test shows that there are statistically significant differences between one or more of the test item groups and the vehicle control group, a Linear regression (p < 0.05) was performed to test whether there is a significant trend in the induction.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
The animals of the groups treated with 62.5 and 125 mg/kg and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality. Clinical observations were made in the groups treated with 250 mg/kg
Vehicle controls validity:
valid
Negative controls validity:
valid
Remarks:
The vehicle of the study: propylene glycol
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY:
DOSE-RANGE FINDING STUDY:
Selection of an adequate dose-range for the Comet main test was based on a dose-range finding study. One dose group, comprising of 3 males and 3 females, was dosed for two consecutive days (once daily) with the highest concentration of test item that was used for the main study. The observation period after dosing was one to three days. During this period mortality and physical condition were recorded at least once a day (Table 1)
In the dose-range finding test, male and female animals dosed with 250 mg/kg showed treatment related clinical signs (ataxia, lethargy and paralyzed hindlegs). This clearly indicated that the MTD was reached.

RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: Range-finding study.
- Comet Slide Analysis: Comet slides were prepared and analyzed. An overview of the mean Tail Intensity is presented in Table 3 - Table 5. The detailed data is is presented in study report.
No statistically significant increase in the mean Tail Intensity (%) was observed in Liver and Glandular Stomach cells of test item treated male treated animals compared to the vehicle treated animals. A statistically significant increase was observed in Duodenum cells of the animals treated with 62.5 mg/kgcompared to the vehicle control treated animals. However, no significant concentration-related increase was observed, and the results are within the 95% control limits of the negative historical control data range. Therefore, the increase was considered not of biological relevance.
The mean Tail Intensity in Liver, Duodenum and Glandular Stomach cells of vehicle-treated rats was 4.54 ± 0.32% (mean ± SD), 5.88 ± 1.86% (mean ± SD) and 6.33 ± 2.80% in male animals, respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control (Table 25). The positive control EMS induced a significant increase and showed a mean Tail Intensity of 95.72 ± 1.48% (mean ± SD; p<0.001 Students t test), 62.31 ± 4.59% (mean ± SD; p<0.001 Students t test) and 66.46 ± 1.81% (mean ± SD; p<0.001 Students t test) in male animals in Liver, Duodenum and Glandular Stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database (Table 26). Adequate numbers of cells (150 cells per animal) and doses were analyzed, and the highest test dose was the MTD. Hence, all criteria for an acceptable assay were met.

TABLE 1 - Mortality and Toxic Signs in the Dose-range Finding Study:




































































GroupSexAnimal NumberDose mg/kgToxic signs:
Day 1 - post-dose
Toxic signs:
Day 2 - Pre-dose
Toxic signs:
Day 2 - Post-dose
1Male101250LethargyShowed no abnormalitiesAtaxia
Lethargy
Paralyzed hindlegs
1Female102250LethargyShowed no abnormalitiesAtaxia
Lethargy
Paralyzed hindlegs
1Male103250LethargyShowed no abnormalitiesAtaxia
Lethargy
Paralyzed hindlegs
1Male104250Showed no abnormalitiesShowed no abnormalitiesAtaxia
Lethargy
1Female105250Showed no abnormalitiesShowed no abnormalitiesAtaxia
Lethargy
Paralyzed hindlegs
1Female106250LethargyShowed no abnormalitiesAtaxia
Lethargy
Paralyzed hindlegs

 


TABLE 2 - Mortality and Toxic Signs after Treatment in the Main Study
























































































































































































































































GroupSexAnimal numberDose mg/kgToxic signs - Day 1
Post-dose within 2.5 hrs
Toxic signs - Day 2
Pre-dose
Toxic signs - Day 2
Post-dose within
1Male10showed no abnormalitesshowed no abnormalitesshowed no abnormalites
1 Male20showed no abnormalitesshowed no abnormalitesshowed no abnormalites
1 Male30showed no abnormalitesshowed no abnormalitesshowed no abnormalites
1 Male40showed no abnormalitesshowed no abnormalitesshowed no abnormalites
1 Male50showed no abnormalitesshowed no abnormalitesshowed no abnormalites
2 Male662.5showed no abnormalitesshowed no abnormalitesshowed no abnormalites
2 Male762.5showed no abnormalitesshowed no abnormalitesshowed no abnormalites
2 Male862.5showed no abnormalitesshowed no abnormalitesshowed no abnormalites
2 Male962.5showed no abnormalitesshowed no abnormalitesshowed no abnormalites
2 Male1062.5 showed no abnormalites showed no abnormalites showed no abnormalites
3 Male11125 showed no abnormalites showed no abnormaliteshunched posture
3 Male12125 showed no abnormalites showed no abnormalites showed no abnormalites
3 Male13125lethargy showed no abnormalites showed no abnormalite showed no abnormalitess
3 Male14125lethargy showed no abnormalites showed no abnormalites
3 Male15125 showed no abnormalites showed no abnormalites showed no abnormalites
4 Male16250lethargyshowed no abnormaliteslethargy
paralyzed hindlegs
4 Male17250lethargyshowed no abnormaliteslethargy
paralyzed hindlegs
4 Male18250lethargyshowed no abnormalitesconvulsion
lethargy
paralyzed hindlegs
4 Male19250lethargyshowed no abnormaliteslethargy
paralyzed hindlegs
4 Male20250lethargydied-
4 Male21250lethargyshowed no abnormaliteslethargy
paralyzed hindlegs
4 Male22250lethargyshowed no abnormaliteslethargy
paralyzed hindlegs
4 Male23250lethargy lethargy
5 Male24200 EMSshowed no abnormalitesshowed no abnormalitesshowed no abnormalites
5 Male25200 EMSshowed no abnormalitesshowed no abnormalitesshowed no abnormalites
5 Male26200 EMSshowed no abnormalitesshowed no abnormalitesshowed no abnormalites

 


TABLE 3 - Overview Tail Intensity in Liver Cells of Males Rats



































 Tail Intensity (mean %)S.D.
Vehicle Control4.540.32
Test Item 62.5 mg/kg4.280.58
Test Item 125 mg/kg3.861.08
Test Item 250 mg/kg5.260.90
EMS 200 mg/kg95.721.48

 


TABLE 4 - Overview Tail Intensity in Duodenum Cells of Male Rats



































 Tail Intensity (mean %)S.D.
Vehicle Control5.881.86
Test Item 62.5 mg/kg9.633.56
Test Item 125 mg/kg6.661.12
Test Item 250 mg/kg7.751.09
EMS 200 mg/kg62.314.59

TABLE 5 - Overview Tail Intensity in Glandular Stomach Cells of Male Rats



































 Tail Intensity (mean %)S.D.
Vehicle Control6.332.80
Test Item 62.5 mg/kg6.741.71
Test Item 125 mg/kg6.061.59
Test Item 250 mg/kg7.211.01
EMS 200 mg/kg66.461.81
Conclusions:
In conclusion, under the experimental conditions, Toluene-4-sulphonohydrazide has not shown any evidence of causing an increase in DNA strand breaks in Liver, Duodenum and Glandular Stomach cells when sampled approximately 3-4 hours post dosing, of male rats, dosed via oral gavage for two consecutive days up to a dose of 250 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines).
Thus, Toluene-4-sulphonohydrazide was found not to be genotoxic in the Comet assay (OECD 489). The results obtained with the positive control substance EMS confirmed the validity of the test.
Executive summary:

The study procedures described were based on the most recent OECD 489 guideline.


Based on the results of the dose-range finding study test concentrations of 250 mg/kg/day for male animals was selected as maximum dose for the main test (maximum tolerated dose). Since there were no substantial differences in toxicity between sexes only males were used in the main study.


The concentrations analyzed in the formulations of Group 2 (62.5 mg/kg), Group 3 (125 mg/kg) and Group 4 (250 mg/kg) were in agreement with target concentrations (i.e., mean sample concentration results were within or equal to 90-110% of target concentration). No test item was detected in the Group 1 (control group) formulation. The formulations of Group 2 (62.5 mg/kg) and Group 4
(250 mg/kg) were homogeneous (i.e., coefficient of variation ≤ 10%).


 


In the main study male animals were dosed with vehicle (propylene glycol), test item
(at 62.5, 125 and 250 mg/kg body weight) for two consecutive days. A positive control group was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight.


Clinical signs of toxicity were limited to the middle and high dose group and included convulsion, lethargy, hunched posture and paralyzed hindlegs. In the highest dose-group 1 animal was found death on day 2.


 


Approximately 3-4 hours after the last dose the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia tissues were isolated. Single cell suspensions from liver, stomach and duodenum were made followed by Comet slide preparation. The slides were analyzed and the Tail Intensity (%) was assessed.


No statistically significant increase in the mean Tail Intensity (%) was observed in Liver, Duodenum and Glandular Stomach cells of test item treated male treated animals compared to the vehicle treated animals. A statistically significant increase was observed in Duodenum cells of the animals treated with 62.5 mg/kg compared to the vehicle control treated animals. However, no significant concentration-related increase was observed, and the results are within the 95% control limits of the negative historical control data range.  Therefore, the increase was considered not of biological relevance.


The mean Tail Intensity in Liver, Duodenum and Glandular Stomach cells of vehicle-treated rats was 4.54 ± 0.32% (mean ± SD), 5.88 ± 1.86% (mean ± SD) and 6.33 ± 2.80% in male animals, respectively, which is within the 95% control limits of the distribution of the


historical control data for the vehicle control. The positive control EMS induced a significant increase and showed a mean Tail Intensity of 95.72 ± 1.48% (mean ± SD), 62.31 ± 4.59% (mean ± SD) and 66.46 ± 1.81% (mean ± SD) in male animals in Liver, Duodenum and Glandular Stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database. Adequate numbers of cells and doses were analyzed, and the highest test dose was the MTD. Hence, all criteria for an acceptable assay were met. There were no hedgehogs observed in any of the slides.


In conclusion, under the experimental conditions described in this report, Toluene-4-sulphonohydrazide has not shown any evidence of causing an increase in DNA strand breaks in Liver, Duodenum and Glandular Stomach cells when sampled approximately 3-4 hours post dosing, of male rats, dosed via oral gavage for two consecutive days up to a dose of 250 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines). Thus, Toluene-4-sulphonohydrazide was found not to be genotoxic in the Comet assay (OECD 489). The results obtained with the positive control substance EMS confirmed the validity of the test.


 


 


 

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

Additional information

Short description of key information: 


Toluene-4-sulphonohydrazide (TSH) was found to be mutagenic in two bacteria cell assays (OECD 471) with and without metabolic activation (S9 mix) as well as in mammalian cells (OECD 476) without metabolic activation (S9 mix).  Toluene-4-sulphonohydrazide (TSH) did not induce chromosomal aberrations using Chinese hamster ovary (CHO) in accordance to OECD 473 with and without metabolic activation (S9 mix).


 


Toluene-4-sulphonohydrazide (TSH) did not show any evidence of causing an increase in DNA strand breaks in Liver, Duodenum and Glandular Stomach cells when sampled approximately 3-4 hours post dosing, of male rats, dosed via oral gavage for two consecutive days up to a dose of 250 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines). Thus, Toluene-4-sulphonohydrazide (TSH) was found not to be genotoxic (In vivo) in the Comet assay (OECD 489). 


 


Endpoint Conclusion:


Based on available in vitro and in vivo data, it is concluded that Toluene-4-sulphonohydrazide (TSH) does not induce genetic toxicity and thus should not be classified accordingly. 

Justification for classification or non-classification

In summary, based on available in vitro and in vivo data from reliable studies (OECD 471, 473, 476 and 489), it is concluded that Toluene-4-sulphonohydrazide (TSH) does not induce genetic toxicity and thus should not be classified accordingly.