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Description of key information

Two studies are available evaluating the repeated dose toxicity (oral) of Toluene-4-sulphonohydrazide (TSH). 



  • The repeated oral toxicity of TSH was evaluated in a 14-day toxicity study (OECD 407, version 1981) in rodents. TSH was administered to the rats by gavage at doses of 0 (vehicle, corn oil), 0.5, 5, and 50 mg/kg bw/day. A NOAEL < 50 mg/kg bw/day was established based on decreased GOT levels in both male and female rats at dose-level 50 mg/kg bw/day. However, effects on organ weight were observed in female (decreased spleen weight 0.5 mg/kg bw/day) and male (increased kidney weight 50 mg/kg bw/day), but not underlying pathology was confirmed by histopathology.


 



  • The repeated oral toxicity of TSH was evaluated in a combined repeated dose toxicity study with reproduction/developmental toxicity screening (OECD 422), in rodents (Wistar Han rats). The dose levels used in this study were 0, 4, 10 and 25 mg/kg/day, based on the results of a preliminary dose range finding study. Formulation analyses confirmed that formulations of test item in propylene glycol were prepared accurately and homogenously. The following parameters and endpoints relating to repeated dose toxicity were evaluated in this study: mortality/moribundity, clinical signs, functional observations, body weight and food consumption, clinical pathology, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations (estrous cycle was also measured in the study but are not relevant for repeated dose toxicity). The following parental toxicity was observed:


 



- At 4 mg/kg/day, a non-adverse lower mean alanine and aspartate aminotransferase activity was recorded.



- At 10 mg/kg/day, adverse axonal degeneration of the sciatic nerve was observed for a single female, reduced grip strength of the fore- and hindlegs, occasional lower body weight at the end of post-coitum and during lactation, and lower alanine and aspartate aminotransferase activity and higher inorganic phosphate concentration.



- At 25 mg/kg/day, adverse axonal degeneration of the sciatic nerve and skeletal muscle degeneration with atrophy, clinical signs consisting primarily of abnormal gait and abnormal posture of the hind legs, lower grip strength, delayed static righting reflex, lower motor activity, lower body weights, macroscopic caudal emaciation, lower alanine and aspartate aminotransferase, lower creatinine and higher inorganic phosphate concentration.


 


Non adverse morphologic alterations consisted of hepatocellular hypertrophy with correlating higher weight, and higher heart and kidney weights (both without microscopic correlate). Watery clear contents were recorded in the thoracic cavity of one female each at 10 and 25 mg/kg/day, which was considered not adverse. 


In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAEL) for Toluene-4-sulphonohydrazide was established:


 


Parental oral NOAEL for systemic effects: 4 mg/kg/day. 


 


The NOAEL was based on axonal degeneration of the sciatic nerve in one female at 10 mg/kg/day, reduced grip strength of the fore- and hindlegs and lower alanine and aspartate aminotransferase activity. At 25 mg/kg/day, axonal degeneration of the sciatic nerve occurred with skeletal muscle degeneration and atrophy, with associated findings consisting of macroscopic caudal emaciation, abnormal gait, abnormal posture of the hind legs, lower body weight, reduced grip strength of the fore- and hindlegs, delayed static righting reflex, lower mean motor activity, lower alanine and aspartate aminotransferase activity and higher creatinine concentration.


 


 

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
See attached testing report. None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): No information - see statement below.
- Lot/batch number of test material: 202102001
- Purity, including information on contaminants, isomers, etc.: 99.5%

VEHICLE:
- Propylene glycol (Merck, Darmstadt, Germany)

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 for at least 24 hours at room temperature under normal laboratory light conditions and for at least 8 days in the refrigerator is confirmed over the concentration range 1 to 400 mg/mL (suspensions).

Documentation of the identity, strength, purity, composition, and stability for the test item was provided. The Sponsor has appropriate documentation on file concerning the method of synthesis,
fabrication or derivation of the test, and this information is available to the appropriate regulatory agencies should it be requested.


Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France. Details are documented in raw data and report.
- Number of Males: 40
- Number of Females: 48
- Number of pups Expected: Approx. 480 (40 litters x 12)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males: approximately 10-12 weeks. Females: approximately 12-14 weeks.
- Weight at study initiation: Males: 291 – 340 g. Females: 196 – 247 g
- Fasting period before study: No
- Housing: polycarbonate cages and Makrolon plastic cages.
- Diet (e.g. ad libitum): Ad libitum, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours
- Water (e.g. ad libitum): ad libitum
- Acclimation period: The animals was acclimated to the Test Facility toxicology accommodation for 8 days prior to start of the pretest period (females) or at least 5 days before the
commencement of dosing (males).

Animals was randomly assigned to groups at arrival. Males and females was randomized separately. On arrival and during the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together). During the mating phase, males and females were cohabitated on a 1:1 basis. During the post-mating phase, males were housed in their home cage with a maximum of 5 males/cage. Females were individually housed, continuing in the lactating phase with pups, except during locomotor activity monitoring of the dams.

DETAILS OF FOOD AND WATER QUALITY:
Water: Municipal tap water (Periodic analysis of the water was performed, and results of these
analyses are on file at the Test Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study)
Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany (Results of analysis for nutritional components and environmental contaminants are provided by the Supplier and are on file at the Test Facility. It is considered that there are no known contaminants in the
feed that would interfere with the objectives of the study)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21 ( The values that were outside the targeted range (18-21°C) occurred occasionally for three days with a minimum of 17°C and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study)
- Humidity (%): 46-71 (The value that was outside the targeted range (40-70%) occurred occasionally for one day with a maximum of 71% and was without a noticeable effect on the clinical condition of the animals or on the outcome of the study.)
- Air changes (per hr): > 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12-hours light and 12-hours dark.

IN-LIFE DATES:
Study Initiation data: 6. July 2021
Initiation of Dosing: 9. September 2021
Completion of In-life: 3. November 2021
Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was selected because this is a possible route of human
exposure during manufacture, handling or use of the test item.
Vehicle:
propylene glycol
Remarks:
Merck, Darmstadt, Germany
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- Procedure: Formulations (w/w) homogenized to visually acceptable levels
- Frequency of preparation: At least weekly filled out in daily portions
- Storage conditions: 4°C
- The dosing formulations was removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were prior to the study performed to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: Dose levels (mg/kg/day) were 0 (vehicle), 4 , 10 and 25.
- Amount of vehicle (if gavage): Dose volume were 4 mL/kg in all groups.
- Lot/batch no. (if required): No information
- Purity: No information

The oral route of administration was selected to ensure systemic exposure to the animals.
The dose levels were selected based on the results of a 10-day dose range finder with oral gavage administration of Toluene-4-sulphonohydrazide in rats (Test Facility Study No.
20285169, see Section 5), and in an attempt to produce graded responses to the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Accuracy: Dose formulation samples were collected for analysis in week 1 of treatment for all dose groups. The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e., mean sample concentration results were within or equal to 85-115% of target concentration). No test item was detected in the Group 1 formulation.
Homogeneity: Dose formulations samples were collected in week of of treatment for groups 2 and 4. The formulations of Groups 2 and 4 were homogeneous (i.e., coefficient of variation ≤ 10%).
Duration of treatment / exposure:
Males: 29
Females: 42-55
Frequency of treatment:
Daily, 7 days each week.
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
4 mg/kg bw/day (nominal)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle (propylene glycol)
No. of animals per sex per dose:
10 females and 10 males in each dose group of main study.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 10-day dose range finder with oral gavage administration of Toluene-4-sulphonohydrazide in rats, and in an attempt to produce graded responses to the test item.
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: F0-males Yes (overnight with a maximum of 24 hours). F0-females: No
- Rationale for selecting satellite groups: No satellite groups

DOSE RANGE FINDER
A Dose Range Finder was conducted to select dose levels for the Main study and to determine the peak effect of occurrence of clinical signs after dosing (For data collection in the Dose Range Finder, an additional Test Facility Reference No. was generated (No. 20285165). No testing guidelines was applicable as the dose range finder was intended for dose level selection purposes only. The information, procedures and safety instructions was identical to those used during the Main study. The dose levels were selected based on information provided by the Sponsor (Chemical Safety Report, 28/03/2018 - IUCLID 6 v2.0.0). Based on data from a structurally similar compound, the parental NOAEL was expected to be around 10 mg/kg/day, with adverse effects (primarily liver and kidneys as target organs) emerging around 30-50 mg/kg/day.

- Number of animals: Females: 6 (nulliparous and non-pregnant). Males: 0.
- Test system: On 07 Jul 2021, female Crl: WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. Females were 12-13 weeks old and weighed between 204-211 (Group 1), 14-15 weeks old and weighed between 197-222 (Group 2) and 13-14 weeks old and weighed between 177-205 (Group 3) at initiation of dosing. On arrival and following assignment to groups at random at the discretion of the biotechnician, animals were group housed (up to 3 animals of the same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm). At study assignment, each animal was identified using earmark and tailmark.

The actual daily mean temperature during the study period was 17 to 21°C with an actual daily mean relative humidity of 52 to 69%. The values that were outside the targeted temperature range occurred for 2 days with a minimum of 17°C and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study.

The dose levels used was: 20, 30 and 50 mg/kg bw/day

Dose concentration: 4, 6 and 10 mg/mL

Number of females: 3 per dose level









Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes / No / Not specified
- Time schedule: At least three time daily: at 0-15 minutes, 1 hour (± 15 minutes) and 3 hours (± 30 minutes) after dosing.
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily, up to the day prior to necropsy.
(These clinical observations was least be conducted at no specific time point, but within a similar time period after dosing for the respective animals).

BODY WEIGHT: Yes
- Time schedule for examinations: On Day 1 of treatment (prior to dosing) and weekly thereafter.
Mated females: on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7,
and 13.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Measured weekly, except for males and females which are housed together for mating and for females without evidence of mating. Mated females: on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Males yes (overnight with maximum of 24 hours). Female no.
- How many animals: All
- Parameters were examined: White blood cells (WBC); Reticulocytes (absolute); Neutrophils (absolute); Red blood cell distribution (RDW); Lymphocytes (absolute); Hemoglobin; Monocytes (absolute); Hematocrit; Eosinophils (absolute); Mean corpuscular volume (MCV); Basophils (absolute); Mean corpuscular hemoglobin (MCH); Large unstained cells (LUC) (absolute);
Red blood cells (RBC); Mean corpuscular hemoglobin concentration (MCHC); and Platelets

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Animals fasted: Males yes (overnight with maximum of 24 hours). Female no.
- How many animals: All
- Parameters that were examined: Alanine aminotransferase (ALT); Creatinine; Aspartate aminotransferase (AST); Glucose; Alkaline phosphatase (ALP); Cholesterol; Total protein; Sodium;
Albumin; Potassium; Total bilirubin; Chloride; Bile acids; Calcium; Urea; and Inorganic phosphate (Inorg. phos)

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: On the day of scheduled necropsy
- Animals fasted: Males yes (overnight with maximum of 24 hours). Female no.
- How many animals: All

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 5 selected males was tested once during Week 4 of treatment and 5 selected females was tested once during the last week of lactation (i.e. PND 6-13)-
These tests was performed after clinical observations (including arena observation, if applicable).
- Dose groups that were examined: 1,2,3,4
- Battery of functions tested: hearing ability, pupillary reflex and static righting reflex (score
0 = normal/present, score 1 = abnormal/absent); fore- and hind-limb grip strength recorded as the mean of three measurements , using a grip strength meter (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands); locomotor activity (recording period: 1 hour under normal
laboratory light conditions) was tested using the Kinder Scientific Motor Monitor System LLC, Poway, USA. Total movements and ambulations was reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but
also smaller or finer movements like grooming, weaving or movements of the head.

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, evaluations performed in accordance to the OECD 422 study guideline (see below tables)

HISTOPATHOLOGY: Yes, evaluations performed in accordance to the OECD 422 study guideline (see below tables)
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons
were conducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions were analyzed according to sex and
occasion. Descriptive statistics number, mean and standard deviation were reported
whenever possible. Values were expressed as a percentage of predose or control
values when deemed appropriate. Inferential statistics were performed according to the
matrix below when possible, but excluded semi-quantitative data, and any group with less
than 3 observations.
The following pairwise comparisons was made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test item-related clinical signs were noted at 25 mg/kg/day from Week 4 of dosing onwards
and included an abnormal gait (up to seven males and all females) and abnormal posture of
the hind legs (all females). At lower incidence, a hunched posture (females only), piloerection
(both sexes) and gasping (females only) were observed. In addition, one female presented
with tremors at slight to moderate degree during Weeks 4-6 of dosing. Hunched posture was
also recorded for one female at 10 mg/kg/day in Week 7 of dosing.
No findings were noted during the weekly arena observations in this study.
Salivation was observed at increasing incidence at 4 (males only), 10 and 25 mg/kg/day from
Week 2 of dosing onwards. This sign was considered not toxicologically relevant, taking into
account the nature and minor severity of the effect and its time of occurrence (i.e. after
dosing). It was considered to be a physiological response rather than a sign of systemic
toxicity.
Other clinical signs recorded during dosing were considered unrelated to treatment with the
test item. Deep respiration and gasping recorded for single animals at 4 and 10 mg/kg/day
occurred in the absence of a dose-related trend and were therefore considered unrelated to
treatment with the test item. Stereotype behavior was observed in several animals at 10
mg/kg/day on Day 43 of treatment. This finding was considered unrelated to treatment, since
it occurred on a single day of treatment only and no trend was apparent regarding dose and
duration of treatment. Remaining findings included scabs, alopecia, piloerection, dark feces, a
small eye and exophthalmos. These findings were either limited to control animals or
occurred within the range of background findings to be expected for rats of this age and strain
which are housed and treated under the conditions in this study and were therefore considered
not to be related to treatment with the test item.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 25 mg/kg/day, males showed a slightly lower mean body weight and body weight gain on
Day 15 of mating only. As a result, mean body weight was 6% lower compared to control at
the end of the dosing period. Females at this dose level showed a lower mean body weight
from Day 4 post-coitum onwards, extending into the lactation period. This resulted in a mean
body weight that was 16 or 15% lower at the end of the post-coitum and lactation period,
respectively. Mean body weight gain of these females was also lower throughout the postcoitum period, and on Day 7 of lactation only.
At 10 mg/kg/day, mean body weight was slightly lower on Day 20 post-coitum, with weight
gain being slightly lower on Lactation Day 7 only.
At 4 mg/kg/day, no test item-related changes in body weight and body weight gain were
recorded.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was considered unaffected by
treatment with the test item.
Any changes in absolute and relative food consumption for females at 25 mg/kg/day during
the post-coitum period were considered to be unrelated to treatment with the test item, since
no trend was apparent regarding duration of treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological parameters of treated rats were considered not to be affected by treatment. In males, a higher reticulocyte count (RETIC; 1.29x of control) was noted at 25 mg/kg/day.
This was considered unrelated to treatment with the test item, since a comparable increase
was observed at 10 mg/kg/day and no changes in correlating hematology parameters were
noted.
Any other changes in hematology parameters, regardless of reaching statistical significance,
were considered to be unrelated to treatment with the test item as these occurred in the
absence of a dose-related trend or were caused by a general overlap and variability in
individual values.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical biochemistry parameters were considered affected by treatment with the test item at
all tested dose levels. The following changes distinguished treated males from control
animals:
• Lower mean alanine aminotransferase (ALT) activity for males at 4, 10 and 25 mg/kg/day (0.77, 0.59 and 0.50x of control, respectively) and for females at 25 mg/kg/day (0.52x of control).
• Lower mean aspartate aminotransferase (AST) activity for males and females at 4, 10 and 25 mg/kg/day (0.74, 0.49 and 0.33x of control, respectively for males, and 0.68, 0.57 and 0.45x of control, respectively for females).
• Lower mean creatinine (CREAT) concentration for males and females at 25 mg/kg/day (0.85x and 0.82x of control, respectively).
• Higher mean inorganic phosphate (PHOS) concentration at 10 and 25 mg/kg/day (1.13
and 1.26x of control, respectively).

Any other changes in clinical biochemistry parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend and were
caused by a general overlap and variability in individual values.
Endocrine findings:
no effects observed
Description (incidence and severity):
Thyroid hormone analyses: Serum levels of T4 in F0-males were considered unaffected by treatment with the test item.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
At 10 and 25 mg/kg/day, mean grip strength of the fore- and/or hindlegs was lower for both
sexes. At 10 mg/kg/day, mean grip strength of the forelegs was 0.75x and 0.88x of control for
males and females, respectively, and mean grip strength of the hindlegs was 0.86x of control
for males (not statistically significant for any of these means). At 25 mg/kg/day, mean grip
strength of the forelegs was 0.61x and 0.32x of control for males and females, respectively
(not statistically significant for males), and mean grip strength of the hindlegs was 0.33x and
0.21x of control for males and females, respectively.
All examined females at 25 mg/kg/day also had a delayed static righting reflex.
Additionally, females at 25 mg/kg/day had a lower mean motor activity, both in terms of total
movements (0.80x of control, not statistically significant) and ambulations (0.61x of control,
not statistically significant).
Grip strength at 4 mg/kg/day, and motor activity and static righting reflex at 4 and 10
mg/kg/day was considered unaffected by treatment with the test item. Hearing ability and
pupillary reflex were normal in all examined animals.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Higher organ weights (relative to body weights) were noted in the 25 mg/kg/day groups males
and females. Test item-related higher heart weights (relative to body weight) were present in males and
females at 25 mg/kg/day and higher kidney and liver weights (relative to body weight) were
present in males at 25 mg/kg/day. All other statistically significant organ weight changes
(relative weight in females of uterus at 4 mg/kg/day and brain, liver, uterus and kidney at
25 mg/kg/day) were considered to be the result of a lower terminal body weight or did not
show a dose response
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
A test item-related macroscopic caudal emaciation was observed in 8/10 females treated at
25 mg/kg/day. There were two females (No. 70 at 10 mg/kg/day and No. 73 at 25 mg/kg/day)
with watery clear contents in the thoracic cavity. There were no microscopic findings in the thoracic cavity organs that could be correlated to this finding and a test item-relation was
unclear.
The remainder of the recorded macroscopic findings was within the range of background
gross observations encountered in rats of this age and strain
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with Toluene-4-sulphonohydrazide
were noted in the liver, sciatic nerve and skeletal muscle and are summarized in Table 1.
Liver, hepatocellular hypertrophy was present in 3/5 males treated at 25 mg/kg/day at
minimal degree.
Sciatic nerve, axonal degeneration was present in all males treated at 25 mg/kg/day at
moderated degree, a single female treated at 10 mg/kg/day at minimal degree and all females
at 25 mg/kg/day at slight to moderate degree.
Skeletal muscle, degeneration with atrophy was present in all males and females treated at
25 mg/kg/day at minimal to moderate degree.
Skeletal muscle, an increased incidence and severity of inflammatory cell infiltrate was
present in males at 25 mg/kg/day at minimal to slight degree.
There were no other test item-related histologic changes. The remainder of the recorded
microscopic findings were within the range of background pathology encountered in rats of
this age and strain. There was no test item-related alteration in the prevalence, severity, or
histologic character of those incidental tissue alterations.
Key result
Dose descriptor:
NOAEL
Effect level:
4 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
clinical biochemistry
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
10 mg/kg bw/day (nominal)
System:
peripheral nervous system
Organ:
other: sciatic nerve
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
yes

Results form the Dose Range Finder (DRF) study:


 
















































Parameter



50 mg/kg/day



30 mg/kg/day



20 mg/kg/day



Mortality



3/3 animals sacrificed on Day 8 of treatment.



No mortality.



No mortality.



Clinical appearance



In 3/3 animals, abnormal breathing sounds, an erected fur, a hunched posture, a splayed foot of the left and right hindpaw and a limited usage (at a slight to severe degree) of the left and right hindlimb were observed on Days 7 and/or 8.


In 2/3 animals, labored breathing, severely uncoordinated movements a lack of or impaired righting reflex, paresis of the forepaws and hindlimbs and ataxia were noted on Days 7 and/or 8.


Symptoms were noted directly after dosing, after 1 hour and/or 3 hours after dosing.



In 3/3 animals, an erected fur was noted on Days 16, 17 and/or 21, an abnormal gait between Days 18-21 and a hunched posture on Day 21.


In 2/3 animals, low carriage was observed between Days 18-20 and splayed foot of the left and right hindpaw and salivation (at a slight to moderate degree) on Days 18, 20 and/or 21.


Symptoms were noted either directly after dosing or at 1 and/or 3 hours post-dose.



No signs noted.



Body weight



Severe weight loss (12-13%) in 2/3 animals between Days 5-8.


Slight weight loss (1-2%) in 2/3 animals between Days 1-5 and 1/3 animals between Days 5-8.



Slight weight loss (up to 4%) in 1/3 animals between Days 10-15, 2/3 animals between Days 15-18 and 3/3 animals between Days 18-21.



Slight weight loss (up to 2%) in 1/3 animals between Days 10-15 and in 2/3 animals between Days 18-21.



Food consumption



Lower during Days 1-5.



(Slightly) lower from Day 10 onwards.



Normal.



Macroscopic examination



No macroscopic findings noted.



No macroscopic findings noted.



In 1/3 animals, a dark, tan focus on the clitoral gland was noted.



Organ weights



-



Kidney and liver weights were considered normal.



Kidney and liver weights were considered normal.



-= not applicable


 


Based on the results of this DRF study, selected dose levels for the Main study were 4, 10 and 25 mg/kg/day. A high dose level of 25 mg/kg/day was selected since dose levels of 50 and 30 mg/kg/day were considered to exceed the maximum tolerated dose, taking into account the longer treatment duration and possible higher sensitivity of pregnant females to treatment in an OECD 422 study. Since no clear peak effect of occurrence of clinical signs was observed in the DRF, clinical observations were conducted and functional observations were started in the Main study after dosing at no specific time point, but within a similar time period after dosing for the respective animals.


 


 


Key results from the main study:


 


Table 1 Experimental Design































































Group No.



Test Item Id.



Dose Level


(mg/kg/day)



Dose Volume (mL/kg)



Dose Concentration (mg/mL)



Number of Animals



Animal Numbers



Males



Females



Males



Females



1



-



0 (Vehicle)



4



0



10



10



01-10



41-50



2



Toluene-4-sulphonohydra
zide



4



4



1



10



10



11-20



51-60



3



10



4



2,5



10



10



21-30



61-70



4



25



4



6,25



10



10



31-40



71-80



 


 


  Table 2 Mean percent organ weight differences from control group




































































































































































 



Males



Females



Dose level (mg/kg/day):



4



10



25



4



10



25



 



 



 



 



 



 



 



BODY WEIGHT



 



 



 



 



 



 



               Absolute



5



2



-6*



1



-5



-17**



 



 



 



 



 



 



 



HEART



 



 



 



 



 



 



               Absolute



0



3



11



2



0



13



               Relative to body weight



-7



-4



19**



6



7



35**



 



 



 



 



 



 



 



LIVER



 



 



 



 



 



 



               Absolute



5



9



5



-4



-6



-9



               Relative to body weight



-1



1



13*



0



2



10**



 



 



 



 



 



 



 



KIDNEY



 



 



 



 



 



 



               Absolute



6



7



10



-2



-3



-1



               Relative to body weight



0



0



18**



1



4



19**



 



 



 



 



 



 



 



  *: P≤0.05, **: P≤0.01


 


Table 3 Summary test-item related microscopic findings


















































































































































































































































 



Males



Females



Dose level (mg/kg/day):



0



4



10



25



0



4



10



25



 



 



 



 



 



 



 



 



 



LIVER a



5



5



5



5



6



1



-



5



    Hepatocellular hypertrophy



 



 



 



 



 



 



 



 



       Minimal



-



-



-



3



-



-



-



-



 



 



 



 



 



 



 



 



 



SKELETAL MUSCLE a



5



5



5



5



5



5



5



5



    Inflammatory cell infiltrate



 



 



 



 



 



 



 



 



       Minimal



1



-



-



3



1



-



-



2



       Slight



-



-



-



2



-



-



-



-



 



 



 



 



 



 



 



 



 



    Degeneration with atrophy



 



 



 



 



 



 



 



 



       Minimal



-



-



-



1



-



-



-



1



       Slight



-



-



-



3



-



-



-



2



       Moderate



-



-



-



1



-



-



-



2



 



 



 



 



 



 



 



 



 



SCIATIC NERVE a



5



5



5



5



5



5



5



5



    Axonal degeneration



 



 



 



 



 



 



 



 



       Minimal



-



-



-



-



-



-



1



-



       Slight



-



-



-



-



-



-



-



1



       Moderate 



-



-



-



5



-



-



-



4



   a  =  Number of tissues examined from each group.


 


Liver, hepatocellular hypertrophy was present in 3/5 males treated at 25 mg/kg/day at minimal degree.



Sciatic nerve, axonal degeneration was present in all males treated at 25 mg/kg/day at moderated degree, a single female treated at 10 mg/kg/day at minimal degree and all females at 25 mg/kg/day at slight to moderate degree.



Skeletal muscle, degeneration with atrophy was present in all males and females treated at 25 mg/kg/day at minimal to moderate degree.



Skeletal muscle, an increased incidence and severity of inflammatory cell infiltrate was present in males at 25 mg/kg/day at minimal to slight degree.



There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. 


 


Table 4 Males - Functional Observations Summary






























































































































 



 



 


GROUP 1 CONTROL



 


GROUP 2


4 MG/KG/DAY



 


GROUP 3


10 MG/KG/DAY



 


GROUP 4


25 MG/KG/DAY



 


END OF TREATMENT


HEARING



 


MEDIAN



 


0



 


0



 


0



 


0



SCORE 0/1



N



5



5



5



5



PUPIL L



MEDIAN



0



0



0



0



SCORE 0/1



N



5



5



5



5



PUPIL R



MEDIAN



0



0



0



0



SCORE 0/1



N



5



5



5



5



STATIC R



MEDIAN



0



0



0



0



SCORE 0/1



N



5



5



5



5



GRIP FORE



MEAN



975



932



735



593



GRAM



ST.DEV



244



247



279



215



 



N



5



5



5



5



GRIP HIND



MEAN



680



684



585



221 **



GRAM



ST.DEV



107



72



129



59



 



N



5



5



5



5



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


+/++ Steel-test significant at 5% (+) or 1% (++) level


 


Table 5 Female - Functional Observations Summary


































































































































FEMALES



 



 



 



 


GROUP 1 CONTROL



 


GROUP 2


4 MG/KG/DAY



 


GROUP 3


10 MG/KG/DAY



 


GROUP 4


25 MG/KG/DAY



 


END OF TREATMENT


HEARING



 


MEDIAN



 


0



 


0



 


0



 


0



SCORE 0/1



N



5



5



5



5



PUPIL L



MEDIAN



0



0



0



0



SCORE 0/1



N



5



5



5



5



PUPIL R



MEDIAN



0



0



0



0



SCORE 0/1



N



5



5



5



5



STATIC R



MEDIAN



0



0



0



1 +



SCORE 0/1



N



5



5



5



5



GRIP FORE



MEAN



1344



1381



1181



433 **



GRAM



ST.DEV



302



299



95



87



 



N



5



5



5



5



GRIP HIND



MEAN



480



463



443



101 **



GRAM



ST.DEV



83



107



103



34



 



N



5



5



5



5



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


+/++ Steel-test significant at 5% (+) or 1% (++) level


 


 


Table 6 Male - Clinical Chemistry Summary








































































































































































































































































Sex: Male



Reporting Biochemistry



ALT (U/L)


[G]



AST (U/L)


[G1]



ALP (U/L)


[G]



TPROT


 


(g/L)


 


[G]



ALB


 


(g/L)


 


[G]



TBIL


(umol/L) [G1]



BILEAC


(umol/L) [G]



UREA


(mmol/L) [G]



CREAT


(umol/L) [G]



GLUC


(mmol/L) [G]



CHOL


(mmol/L) [G]



NA


(mmol/L) [G1]



0



Mean



60.6



86.5



113.7



63.44



39.44



2.12



46.99



6.85



28.2



7.904



1.546



144.8



mg/kg/day



SD



10.5



14.8



14.4



2.10



1.68



0.22



18.13



0.66



1.9



1.037



0.292



0.4



Group 1



N



5



5



5



5



5



5



5



5



5



5



5



5



4



Mean



46.9 *



63.6



115.4



63.34



39.54



1.94



36.26



7.60



28.2



8.428



1.578



144.2



mg/kg/day



SD



4.7



5.7



14.8



2.12



1.66



0.15



17.16



0.80



1.3



1.543



0.169



1.3



Group 2



N



5



5



5



5



5



5



5



5



5



5



5



5



 



tCtrl



0.77



0.74



1.01



1.00



1.00



0.92



0.77



1.11



1.00



1.07



1.02



1.00



10



Mean



35.5 **



42.6 *



130.5



63.62



39.30



2.02



32.34



7.22



28.7



8.584



1.760



144.4



mg/kg/day



SD



8.8



5.4



25.5



1.13



0.84



0.38



12.49



0.63



2.9



0.863



0.275



0.5



Group 3



N



5



5



5



5



5



5



5



5



5



5



5



5



 



tCtrl



0.59



0.49



1.15



1.00



1.00



0.95



0.69



1.05



1.02



1.09



1.14



1.00



25



Mean



30.1 **



28.2 **



91.5



62.98



38.78



1.96



44.88



8.13



23.9 *



7.964



1.802



144.4



mg/kg/day



SD



5.8



3.6



21.3



2.09



0.84



0.09



28.26



0.89



2.7



0.674



0.040



0.9



Group 4



N



5



5



5



5



5



5



5



5



5



5



5



5



 



tCtrl



0.50



0.33



0.80



0.99



0.98



0.92



0.96



1.19



0.85



1.01



1.17



1.00


























































































































































Sex: Male



Reporting Biochemistry



Reporting S



K


(mmol/L) [G]



CL


(mmol/L) [G]



CA


(mmol/L) [G]



PHOS


(mmol/L) [G]



T4


(ng/mL) [G]



0



Mean



3.92



104.0



2.544



2.010



58.12



mg/kg/day



SD



0.13



1.2



0.022



0.166



12.25



Group 1



N



5



5



5



5



10



4



Mean



4.11



102.2



2.568



2.026



49.22



mg/kg/day



SD



0.22



1.3



0.046



0.056



7.01



Group 2



N



5



5



5



5



10



 



tCtrl



1.05



0.98



1.01



1.01



0.85



10



Mean



3.88



102.6



2.570



2.276 **



53.63



mg/kg/day



SD



0.09



0.9



0.038



0.048



10.72



Group 3



N



5



5



5



5



10



 



tCtrl



0.99



0.99



1.01



1.13



0.92



25



Mean



4.07



102.2



2.664



2.540 **



50.55



mg/kg/day



SD



0.17



1.5



0.170



0.087



10.36



Group 4



N



5



5



5



5



10



 



tCtrl



1.04



0.98



1.05



1.26



0.87



[G] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01


[G1] - Kruskal-Wallis & Dunn: * = p ≤ 0.05; ** = p ≤ 0.01


 


Table 6 Female - Clinical Chemistry Summary








































































































































































































































































Sex: Female



Reporting Biochemistry



ALT (U/L)


[G]



AST (U/L)


[G]



ALP (U/L)


[G]



TPROT


 


(g/L)


 


[G]



ALB


 


(g/L)


 


[G]



TBIL


(umol/L) [G]



BILEAC


(umol/L) [G1]



UREA


(mmol/L) [G]



CREAT


(umol/L) [G]



GLUC


(mmol/L) [G]



CHOL


(mmol/L) [G]



NA


(mmol/L) [G]



0



Mean



170.3



122.0



252.7



54.84



35.64



2.30



12.49



9.69



20.7



6.342



2.122



143.0



mg/kg/day



SD



57.1



25.6



125.9



1.51



1.53



0.28



4.63



0.53



2.7



0.717



0.387



1.6



Group 1



N



5



5



5



5



5



5



5



5



5



5



5



5



4



Mean



123.3



82.8 **



199.9



53.10



34.84



2.22



40.58



10.28



21.2



6.116



1.978



142.2



mg/kg/day



SD



27.1



11.7



79.8



2.49



1.49



0.33



37.73



0.93



2.3



1.714



0.112



1.9



Group 2



N



5



5



5



5



5



5



5



5



5



5



5



5



 



tCtrl



0.72



0.68



0.79



0.97



0.98



0.97



3.25



1.06



1.03



0.96



0.93



0.99



10



Mean



129.6



69.1 **



152.5



54.54



35.56



1.92



14.10



9.84



21.0



6.698



2.158



141.8



mg/kg/day



SD



29.4



10.3



53.2



2.70



1.99



0.27



6.44



0.59



2.5



0.800



0.351



0.8



Group 3



N



5



5



5



5



5



5



5



5



5



5



5



5



 



tCtrl



0.76



0.57



0.60



0.99



1.00



0.83



1.13



1.02



1.02



1.06



1.02



0.99



25



Mean



88.4 **



54.5 **



144.3



53.08



34.78



2.16



38.11



9.25



17.0



7.938



2.076



141.8



mg/kg/day



SD



14.8



3.2



80.3



1.85



1.49



0.44



25.75



0.72



1.7



0.637



0.273



1.6



Group 4



N



5



5



5



5



5



5



5



5



5



5



5



5



 



tCtrl



0.52



0.45



0.57



0.97



0.98



0.94



3.05



0.95



0.82



1.25



0.98



0.99



[G] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01


[G1] - Kruskal-Wallis & Dunn: * = p ≤ 0.05; ** = p ≤ 0.01


 


 

Conclusions:
Wistar Han rats were treated with Toluene-4-sulphonohydrazide (TSH) by daily oral gavage at dose levels of 4, 10 and 25 mg/kg/day in accordance to OECD 422. Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the no-observed-adverse-effect levels (NOAEL) for parental toxicity was evaluated to be 4 mg/kg bw/day. The NOAEL was based on axonal degeneration of the sciatic nerve in one female at 10 mg/kg/day, reduced grip strength of the fore- and hindlegs and lower alanine and aspartate aminotransferase activity. At 25 mg/kg/day, axonal degeneration of the sciatic nerve occurred with skeletal muscle degeneration and atrophy, with associated findings consisting of macroscopic caudal emaciation, abnormal gait, abnormal posture of the hind legs, lower body weight, reduced grip strength of the fore- and hindlegs, delayed static righting reflex, lower mean motor activity, lower alanine and aspartate aminotransferase activity and higher creatinine concentration.
Executive summary:

The objectives of this OECD 422 study were to determine the potential repeated dose toxicity as well as reproductive and developmental toxicity of Toluene-4-sulphonohydrazide when given orally by gavage for a minimum of 28 days to Wistar Han rats, evaluating male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated. Data relating to the parental toxicity has has been described. 


 


The dose levels in this study were 0, 4, 10 and 25 mg/kg/day, based on the results of a preliminary dose range finding study. Formulation analyses confirmed that formulations of test item in propylene glycol were prepared accurately and homogenously.


 


The following parameters and end points relting to repeated dose toxicity were evaluated in this study: mortality/moribundity, clinical signs, functional observations, body weight and food consumption, clinical pathology, measurement of thyroid hormone T4 (F0-males), gross
necropsy findings, organ weights and histopathologic examinations (estrous cycle was also measured in the study, but are not relevant for repeated dose toxicty. In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development. PND 14-16 pups: mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4). 


 


Parental toxicity observed:
At 4 mg/kg/day, a non-adverse lower mean alanine and aspartate aminotransferase activity was recorded.


 



At 10 mg/kg/day, adverse axonal degeneration of the sciatic nerve was observed for a single female and in 5/5 females and 5/5 males at 25 mg/kg bw/day. Further, at 10 mg/kg bw/day reduced grip strength of the fore- and hindlegs, occasional lower body weight at the end of post-coitum and during lactation, and lower alanine and aspartate aminotransferase activity and higher inorganic phosphate concentration.


 



At 25 mg/kg/day, adverse axonal degeneration of the sciatic nerve and skeletal muscle degeneration with atrophy, clinical signs consisting primarily of abnormal gait and abnormal posture of the hind legs, lower grip strength, delayed static righting reflex, lower motor activity, lower body weights, macroscopic caudal emaciation, lower alanine and aspartate aminotransferase, lower creatinine and higher inorganic phosphate concentration.


 


Non adverse morphologic alterations consisted of hepatocellular hypertrophy with correlating higher weight, and higher heart and kidney weights (both without microscopic correlate). Watery clear contents were recorded in the thoracic cavity of one female each at 10 and 25 mg/kg/day, which was considered not adverse. 


 


In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAEL) for Toluene-4-sulphonohydrazide was established:


 


Parental NOAEL for systemic effects: 4 mg/kg/day. 


 


The NOAEL was based on axonal degeneration of the sciatic nerve in one female at 10 mg/kg/day, reduced grip strength of the fore- and hindlegs and lower alanine and aspartate aminotransferase activity. At 25 mg/kg/day, axonal degeneration of the sciatic nerve occurred with skeletal muscle degeneration and atrophy, with associated findings consisting of macroscopic caudal emaciation, abnormal gait, abnormal posture of the hind legs, lower body weight, reduced grip strength of the fore- and hindlegs, delayed static righting reflex, lower mean motor activity, lower alanine and aspartate aminotransferase activity and higher creatinine concentration.


 


The severe effects observed fulfills the criteria for a STOT RE 1 classification as the dose levels are below the criteria cut-off value of 30 mg/kg/day (oral, rat) for a subacute study. 


 


 


Overall, the current data indicate that a classification with STOT RE 1, H373 (Neuromuscular system) is warranted for TSH.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
4 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch score 1. The study was performed in accordance with the OCED guideline 422 and according to GLP standard.
System:
other: Neuromuscular system
Organ:
other: Neuromuscular tissue

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The repeated oral toxicity of TSH was evaluated in 2 oral studies:


 


- From an oral 407 study, a NOAEL < 50 mg/kg bw/day was established based on decreased GOT levels in both male and female rats at dose-level 50 mg/kg bw/day. However, effects on organ weight were observed in female (decreased spleen weight 0.5 mg/kg bw/day) and male (increased kidney weight 50 mg/kg bw/day), but not underlying pathology was confirmed by histopathology.


 


- From an OECD 422 study, the parental oral NOAEL for systemic effects ws concluded to be 4 mg/kg/day. The NOAEL was based on axonal degeneration of the sciatic nerve in one female at 10 mg/kg/day, reduced grip strength of the fore- and hindlegs and lower alanine and aspartate aminotransferase activity. At 25 mg/kg/day, axonal degeneration of the sciatic nerve occurred with skeletal muscle degeneration and atrophy, with associated findings consisting of macroscopic caudal emaciation, abnormal gait, abnormal posture of the hind legs, lower body weight, reduced grip strength of the fore- and hindlegs, delayed static righting reflex, lower mean motor activity, lower alanine and aspartate aminotransferase activity and higher creatinine concentration.


 


Based on the available experimental data neuromuscular system are considered the most sensitive organ. From the overall database a NOAEL of 4 mg/kg bw/day for systemic effects on the neuromuscular system was established from and OECD 422 study. 


 


No data available for inhalational or dermal route of exposure.


 


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:


Based on the available experimental data the neuromuscular system are considered the most sensitive organ. From the overall database (OECD 407, 422) a NOAEL of 4 mg/kg bw/day for systemic effects on the neuromuscular system can be established from the OECD 422 study.



Repeated dose toxicity: via oral route - systemic effects (target organ):


Neuromuscular system

Justification for classification or non-classification

The severe effects on the neuromuscular system were identified from an OECD 422 study, where the parental oral NOAEL for systemic effects was concluded to be 4 mg/kg/day. The NOAEL was based on axonal degeneration of the sciatic nerve in one female at 10 mg/kg/day, reduced grip strength of the fore- and hindlegs and lower alanine and aspartate aminotransferase activity. At 25 mg/kg/day, axonal degeneration of the sciatic nerve occurred with skeletal muscle degeneration and atrophy, with associated findings consisting of macroscopic caudal emaciation, abnormal gait, abnormal posture of the hind legs, lower body weight, reduced grip strength of the fore- and hindlegs, delayed static righting reflex, lower mean motor activity, lower alanine and aspartate aminotransferase activity and higher creatinine concentration. The severe effects observed fulfills the criteria for a STOT RE 1 classification as the dose levels are below the criteria cut-off value of 30 mg/kg/day (oral, rat) for a subacute study. 


 


Overall, the current data indicate that a classification with STOT RE 1, H373 (Neuromuscular system) is warranted for TSH.