Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
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 (incl. QA statement)
Limit test:
no
Justification for study design:
NA

Test material

Constituent 1
Chemical structure
Reference substance name:
Toluene-4-sulphonohydrazide
EC Number:
216-407-3
EC Name:
Toluene-4-sulphonohydrazide
Cas Number:
1576-35-8
Molecular formula:
C7H10N2O2S
IUPAC Name:
toluene-4-sulphonohydrazide
Test material form:
solid: particulate/powder
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).

The Sponsor provided a documentation of the identity, strength, purity, composition, and stability for the test item.
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.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar rat strain (Crl:WI(Han) rats) from Charles River Deutschland, Sulzfeld, Germany.

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
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. A total of 40 females will be selected at randomization before initiation of the pretest phase. Each selected female classified as not having regular estrous cycles during the pretest phase will be replaced before initiation of dosing by one of the 8 additional females having regular estrous cycles, if feasible. A total of 40 females with regular estrous cycles will continue in the study. The supernumerary females will then be removed from the study, and their estrous cycle results will be kept in the raw data but will not be reported.
- 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: 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) in polycarbonate cages (Makrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Makrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Makrolon plastic
cages (MIII type, height 18 cm). (Pups were housed with the dam,
except during locomotor activity monitoring of the dams.)
During locomotor activity monitoring, F0-animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
Cages, unless otherwise described, all contained sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- 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.

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
Merck, Darmstadt, Germany
Details on 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.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Maximum of 14 consecutive days
- Proof of pregnancy: sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day will be designated Day 0 post-coitum. Once mating has occurred, the males and females will be separated
- A maximum of 14 days will be allowed for mating, after which females who have not shown evidence of mating will be separated from their males.
- After successful mating each pregnant female was caged (how): Individually housed in Makrolon plastic cages (MIII type, height 18 cm).
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:
Males: 7 days a week for a minimum of 28 days, including at least 2 weeks of treatment prior to mating and during the mating period up to and including the day before scheduled necropsy.
Females: 7 days a week for at least 14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy.
Females were not be dosed during littering.
Doses / concentrationsopen allclose all
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


Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- 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. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes. Water consumption was monitored by visual inspection of the water bottles. If inter group
differences were noted, consumption was assessed by weight
Oestrous cyclicity (parental animals):
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation is observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period.
End of Treatment - on the day of necropsy, a vaginal lavage wase taken to determine the stage of estrus. This was done for all females, except for females that had to be euthanized in extremis or
die spontaneously.
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by serial vaginal lavage procedures.

Sperm parameters (parental animals):
Parameters examined in [P] male parental generations:
Testes were evaluated to assess the progression of stages of the spermatogenic cycle, cell associations, and proportions expected to be present during spermatogenesis along with assessment of interstitial and supporting cell types (Leydig cells, macrophages, vasculature, and rete testis). Any cell- or stage-specificity of testicular findings were noted.
Litter observations:
STANDARDISATION OF LITTERS
To reduce variability among the litters, eight pups from each litter of equal sex distribution (if possible) were be selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
Mortality, Clinical Observations, Individual Body Weights, Sex, Anogenital Distance, Areola/Nipple Retention, abnormalities (particular attention was paid to the external reproductive genitals to examine signs of altered development), thyroid hormone (PND 4 at culling and PND 14-16), and the thyroid was collected from two pups per litter.


GROSS EXAMINATION OF DEAD PUPS:
Recognizable fetuses of females that died spontaneously or was euthanized in extremis was examined externally and sexed (both externally and internally, if possible).
Pups that died or was euthanized before scheduled termination was also examined externally and sexed (both externally and internally, if possible). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death was evaluated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
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 post-coitum period, and on Day 7 of lactation only.
At 10 mg/kg/day, mean body weight in females 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):
no effects observed
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:
no effects observed
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.
Coagulation parameters of treated rats were considered not to be affected by treatment with the test item.
The slightly shortened prothrombin time (PT) in males at 25 mg/kg/day was considered unrelated to administration with the test item due to the minimal magnitude of the change (0.95x of control) and since the opposite effect (i.e., a prolonged PT) would be expected in case of target organ toxicity.
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:
not examined
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.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
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. 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.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Serum levels of T4 in F0 males were considered unaffected by treatment with the test item.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item. All females had regular cycles of 4 or 5 days.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
One couple of the control group and one couple of the 25 mg/kg/day group did not produce offspring, and one couple of the 25 mg/kg/day group was not pregnant. In addition, there was one female of the 10 mg/kg/day group with total litter loss.
No abnormalities were seen in the reproductive organs (or mammary gland of Female No. 68 with total litter loss), which could account for their lack of (healthy) offspring.
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Reproductive performance:
no effects observed
Description (incidence and severity):
One couple of the control group and one couple of the 25 mg/kg/day group did not produce offspring, and one couple of the 25 mg/kg/day group was not pregnant. In addition, there was one female of the 10 mg/kg/day group with total litter loss.
No abnormalities were seen in the reproductive organs (or mammary gland of Female No. 68 with total litter loss), which could account for their lack of (healthy) offspring.
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Details on results (P0)

Mating index was not affected by treatment with the test item. All females showed evidence of mating.


Precoital time was not affected by treatment with the test item.
All females showed evidence of mating within 5 days. For one control female (No. 50) the precoital time could not be determined as mating for this animal was only confirmed indirectly by the observation of an implantation site in the uterus at necropsy.

Number of implantation sites was unaffected by treatment with the test item.
One control female (No. 50) and one female at 25 mg/kg/day (No. 79) had a single implantation site only. As there was no dose-related trend, this finding did not indicate a relation with treatment with the test item.

Fertility index was considered not to be affected by treatment with the test item. The fertility indices were 100% for the control, 4 and 10 mg/kg/day groups and 90% for the 25 mg/kg/day group.
One female at 25 mg/kg/day (No. 76) was not pregnant. As this isolated incidence, the non-pregnancy of this female was considered not to be related to treatment with the test item.





Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
> 25 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment with the test item.
The single pup of Female No. 68 (10 mg/kg/day) that was sacrificed in extremis on PND 7, was noted dehydrated from PND 4 onwards. At last litter check, no milk was present in its stomach.

Other clinical signs (i.e., blue discoloration of the cervical region, black discoloration of the snout, alopecia and scabs) occurred incidentally and in the absence of a dose related trend, and remained within the range considered normal for pups of this age. Therefore, these findings were considered not to be related to treatment with the test item.

No clinical signs were observed for the pups that were found dead or missing
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 25 mg/kg/day, mean body weight of male and female pups was lower from PND 1 onwards (reaching statistical significance for females and both sexes combined on PND 13 only, when mean body weight for both sexes combined was 9% lower).
At 10 mg/kg/day, mean body weight of male and female pups was lower from PND 1 onwards (not statistically significant), being essentially similar to mean weights recorded at 25 mg/kg/day. However, mean weights at 10 mg/kg/day were essentially similar to control mean values on PND 13.
It should be noted that the more pronounced lower mean female pup body weight of 10% on PND 7 (not statistically significant) was considered to be at least in part the result of a very low body weight for the single pup of Litter No. 68 (10 mg/kg/day). After exclusion of this pup from the calculation of the mean body weight, mean female pup body weight was 5% lower on PND 7.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item.
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
Sex ratio was considered not to be affected by treatment with the test item.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item.
Normalized anogenital distance in males was higher at 25 mg/kg/day when compared with concurrent controls. As the opposite effect would be expected in case of target organ toxicity, this higher normalized anogenital distance was considered to be unrelated to treatment with the test item.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Areola/nipple retention was considered unaffected by treatment with the test item.
For one pup at 4 mg/kg/day (Pup No. 4 of Litter No. 60) and for one pup at 25 mg/kg/day (Pup No. 4 of Litter No. 72) one or two nipples were observed. The presence of nipples in these pups was considered unrelated to treatment with the test item as it occurred in the absence of a dose-related trend.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment with the test item.
The stomach of the single pup of Litter No. 68 (10 mg/kg/day) sacrificed in extremis on PND 7 due to dehydration was found to have no milk. As this was an isolated finding in the mid dose group only, this was considered unrelated to treatment with the test item.
Remaining gross findings that were unrelated to treatment with the test item included alopecia for pups of a control litter and autolysis for one dead pup at 4 mg/kg/day.
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Gestation index (females with living pups on Day 1 compared to the number of pregnant females) and duration of gestation were considered not to be affected by treatment with the test item.
The gestation indices were 90, 100, 100 and 89% for the control, 4, 10 and 25 mg/kg/day groups, respectively.
One control female (No. 50) and one female at 25 mg/kg/day (No. 79) had implantation sites only. In absence of a dose related trend, this finding did not indicate a relation with treatment with the test item.


No signs of difficult or prolonged parturition were noted among the pregnant females.
For Female No. 50 (control), blood was found in the cage bedding on Day 39 of treatment . However, it could not be derived if this blood was related to delivery of pups as no pups were found. No deficiencies in maternal care were observed.


Litter size was considered not affected by treatment with the test item. Live litter sizes were 11.2, 11.9, 10.2 and 11.1 living pups/litter for the control, 4, 10 and 25 mg/kg/day groups, respectively. The lower mean number of living pups recorded at 10 mg/kg/day (10.2 vs. 11.2 in the control group; not statistically significant) was attributed to the relatively small litters of Female Nos. 65 and 68 (four and one pup/litter, respectively). In absence of a dose-related trend, this was considered unrelated to treatment with the test item.

The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment with the test item. The live birth indices were 96, 99, 100 and 99% for the control, 4, 10 and 25 mg/kg/day groups, respectively. Four pups of the control group (three pups of Litter No. 42 and one pup of Litter No. 43), one pup at 4 mg/kg/day (Litter No. 51) and one pup at 25 mg/kg/day (Litter No. 72) were found dead at first litter check. These dead pups were considered not to be related to treatment with the test item since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.


The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not affected by treatment with the test item. Viability indices were 96% for the control group and 100% for the 4, 10 and 25 mg/kg/day groups. Four pups of the control group were found missing between PND 2 and 4 (two pups in Litter No. 47 on PND 2, and one pup each in Litter Nos. 44 and 49 on PND 3 and 4, respectively). These missing pups were most likely cannibalized. All pups in the test item-treated groups survived until culling on PND 4.


The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not affected by treatment with the test item. The lactation indices were 100% for the control, 4 and 25 mg/kg/day groups, and 99% at 10 mg/kg/day. Female No. 68 (10 mg/kg/day) had a total litter loss on PND 7 when its single pup was sacrificed in extremis.







Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
body weight and weight gain

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
25 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
presumably yes

Any other information on results incl. tables

Key findings:


Table 1 Reproduction data summary


























































































 



Group 1 (control)



Group 2 (4 mg/kg bw/day)



Group 3 (10 mg/kg bw/day)



Group 4 (25 mg/kg bw/day)


 

 


Females paired



 


10



 


10



 


10



 


10


 

Females mated



10



10



10



10


 

Pregnant females



10



10



10



9


 

Females with implantations only



1



0



0



1


 

Females with living pups on Day 1



9



10



10



8


 

Mating index (%)


(Females mated / Females paired) * 100



100



100



100



100


 
 

Fertility index (%)


(Pregnant females / Females mated) * 100



100



100



100



90


 
 

Gestation index (%)


(Females with living pups on Day 1 / Pregnant females) * 100



90



100



100



89


 
 
 

Gestation index (females with living pups on Day 1 compared to the number of pregnant females) and duration of gestation were considered not to be affected by treatment with the test item. The gestation indices were 90, 100, 100 and 89% for the control, 4, 10 and 25 mg/kg/day groups, respectively. One control female (No. 50) and one female at 25 mg/kg/day (No. 79) had implantation sites only. In absence of a dose related trend, this finding did not indicate a relation with treatment with the test item.


 


 


Table 2 Correlation of Histopathology Findings with In-Life Reason for Males that Failed to Sire and Females that Failed to Deliver Healthy Pups.
















































Group



Dose level


mg/kg bw/day



Female/Male nos.



In-Life Reason



Histopathology



1



0



50/10



Implantation sites only



No histopathological correlate



2



4



-



-



-



3



10



68/-



Total litter loss



No histopathological correlate



4



25



76/36



Not pregnant



No histopathological correlate



4



25



79/39



Implantation sites only



No histopathological correlate



One couple of the control group and one couple of the 25 mg/kg/day group did not produce offspring, and one couple of the 25 mg/kg/day group was not pregnant. In addition, there was one female of the 10 mg/kg/day group with total litter loss.


No abnormalities were seen in the reproductive organs (or mammary gland of Female No. 68 with total litter loss), which could account for their lack of (healthy) offspring.


Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.


 


Table 3 Developmental data



















































































 



Group 1 (control)



Group 2 (4 mg/kg bw/day)



Group 3 (10 mg/kg bw/day)



Group 4 (25 mg/kg bw/day)



 


Total number of offspring born



 


105



 


120



 


102



 


90



Total number of uterine implantation sites



114



138



116



107



Number of live offspring on Day 1 after littering



101



119



102



89



Number of live offspring on Day 4 (before culling)



97



119



102



89



Number of live offspring on Day 4 (after culling)



72



79



69



63



Number of live offspring on Day 13 after littering



72



79



68



63



Post-implantation survival index (%)


(Total number of offspring born/Total number of uterine implantation sites) * 100



92



87



88



84



Live birth index (%)


(Number of live offspring on Day 1 after littering/Total number of offspring born) * 100



96



99



100



99



Viability index (%)


(Number of live offspring on Day 4 (before culling)/Number of live offspring on Day 1 after littering)*100



96



100



100



100



Lactation index (%)


(Number of live offspring on Day 13 after littering/Number of live offspring on Day 4 (after culling)) * 100



100



100



99



100



The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item. Post-implantation survival index was 92, 87, 88 and 84% for the control, 4, 10 and 25 mg/kg/day groups, respectively.


 


Table 4 Body weights of pups (g)



















































































































































































































































































































































DAY



SEX



 



GROUP 1 CONTROL



GROUP 2


4 MG/KG/DAY



GROUP 3


10 MG/KG/DAY



GROUP 4


25 MG/KG/DAY



1



M



MEAN



6.5



6.7



6.3



6.2



 



 



ST.DEV.



0.7



0.6



0.5



0.5



 



 



N



9



10



9



8



 



F



MEAN



6.3



6.4



6.0



5.8



 



 



ST.DEV.



0.7



0.6



0.5



0.4



 



 



N



9



10



10



8



 



M+F



MEAN



6.4



6.5



6.2



6.0



 



 



ST.DEV.



0.7



0.6



0.5



0.5



 



 



N



9



10



10



8



4



M



MEAN



9.7



9.8



9.2



9.1



 



 



ST.DEV.



1.1



1.2



1.1



1.0



 



 



N



9



10



9



8



 



F



MEAN



9.4



9.4



8.5



8.6



 



 



ST.DEV.



1.2



1.0



1.6



0.8



 



 



N



9



10



10



8



 



M+F



MEAN



9.5



9.5



8.6



8.8



 



 



ST.DEV.



1.1



1.1



1.5



0.9



 



 



N



9



10



10



8



7



M



MEAN



15.8



16.2



15.3



14.8



 



 



ST.DEV.



1.5



1.6



1.2



1.1



 



 



N



9



10



9



8



 



F



MEAN



15.5



15.6



14.0



14.1



 



 



ST.DEV.



1.6



1.4



3.0



0.9



 



 



N



9



10



10



8



 



M+F



MEAN



15.6



15.9



14.2



14.4



 



 



ST.DEV.



1.5



1.4



3.0



1.1



 



 



N



9



10



10



8



13



M



MEAN



30.2



31.4



29.8



27.8



 



 



ST.DEV.



2.4



2.4



2.3



1.8



 



 



N



9



10



9



8



 



F



MEAN



29.8



30.3



29.2



26.7 *



 



 



ST.DEV.



2.6



2.1



2.4



1.5



 



 



N



9



10



9



8



 



M+F



MEAN



30.0



30.8



29.5



27.2 *



 



 



ST.DEV.



2.5



2.2



2.3



1.8



 



 



N



9



10



9



8



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


At 25 mg/kg/day, slightly lower body weights were recorded for male and female pups from PND 1 onwards, being 9% lower at PND 13 (combined for both sexes). The lower body weights were considered adverse at this early stage in development, also based on the magnitude of the effect recorded at the end of lactation. Lower body weights recorded at 10 mg/kg/day from PND 1 onwards had essentially recovered on PND 13, and were therefore considered not to be adverse.


 


Table 5 Anogenital distance and nipple retention - Male (M) and Fenale (F)






















































































F0-GENERATION - LACTATION



 



 


GROUP 1 CONTROL



 


GROUP 2


4 MG/KG/DAY



 


GROUP 3


10 MG/KG/DAY



 


GROUP 4


25 MG/KG/DAY



anogenital dist M mm



MEAN



2.67



2.70



2.63



2.82



 



ST. DEV



0.14



0.14



0.18



0.15



 



N



9



10



9



8



anogenital dist F mm



MEAN



0.96



1.00



1.03



1.04



 



ST. DEV



0.11



0.11



0.08



0.11



 



N



9



10



10



8



Number of nipples



MEAN



0.00



0.03



0.00



0.07



 



MEDIAN (+)



0.00



0.00



0.00



0.00



 



N



9



9



6



7






























































 



 


GROUP 1 CONTROL



 


GROUP 2


4 MG/KG/DAY



 


GROUP 3


10 MG/KG/DAY



 


GROUP 4


25 MG/KG/DAY



 


PND 1


norm anog dist M



 


MEAN



 


1.43



 


1.44



 


1.43



 


1.53 +



mm



ST.DEV



0.06



0.06



0.09



0.07



 



N



9



10



9



8



norm anog dist F



MEAN



0.52



0.54



0.56



0.58



mm



ST.DEV



0.06



0.06



0.04



0.06



 



N



9



10



10



8



# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level


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


 


Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item. Normalized anogenital distance in males was higher at 25 mg/kg/day when compared with concurrent controls. As the opposite effect would be expected in case of target organ toxicity, this higher normalized anogenital distance was considered to be unrelated to treatment with the test item.


 


 


Table 6 Summary of Thyroid Hormone values - Male and Female














































































Sex: Male



Reporting Special C



T4


(ng/mL) [G]



0


mg/kg/day Group 1



Mean


SD N



62.37


14.68


9



4



Mean



64.78



mg/kg/day



SD



9.10



Group 2



N



10



 



tCtrl



1.04



10



Mean



60.86



mg/kg/day



SD



11.80



Group 3



N



9



 



tCtrl



0.98



25



Mean



70.39



mg/kg/day



SD



11.85



Group 4



N



8



 



tCtrl



1.13















































































Sex: Female



Reporting Special C



T4


(ng/mL) [G]



0


mg/kg/day Group 1



Mean


SD N



60.94


11.74


9



4



Mean



66.60



mg/kg/day



SD



10.20



Group 2



N



10



 



tCtrl



1.09



10



Mean



60.11



mg/kg/day



SD



13.86



Group 3



N



9



 



tCtrl



0.99



25



Mean



64.79



mg/kg/day



SD



9.39



Group 4



N



8



 



tCtrl



1.06



[G] - Anova & Dunnett

Applicant's summary and conclusion

Conclusions:
The objectives of this OECD 422 study were to determine the potential reproductive and developmental toxicity of Toluene-4-sulphonohydrazidewhen 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. The dose levels in this study were selected to be 0, 4, 10, 25 mg/kg/day, based on the results of the Dose Range Finder.
There were no effects on reproduction, therefore NOAEL was concluded to be at least 25 mg/kg/day. For developmental toxicity, NOAEL was concluded to be 10 mg/kg/day (based on lower pup body weights at 25 mg/kg/day). No other developmental effects were seen.

Executive summary:

The objectives of this OECD 422 study were to determine the potential reproductive and developmental toxicity of Toluene-4-sulphonohydrazidewhen 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. The dose levels in this study were selected to be 0, 4, 10, 25 mg/kg/day, based on the results of the Dose Range Finder. The rats of the control group received the vehicle, propylene glycol, alone.


 


No reproductive toxicity was observed up to the highest dose level tested (25 mg/kg bw/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).


 


No developmental toxicity was observed up to the highest dose level tested (25 mg/kg/day). No toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination). 


 


At 25 mg/kg/day, slightly lower body weights were recorded for male and female pups from PND 1 onwards, being 9% lower at PND 13 (combined for both sexes). The lower body weights were considered adverse at this early stage in development, also based on the magnitude of the effect recorded at the end of lactation. Lower body weights recorded at 10 mg/kg/day from PND 1 onwards had essentially recovered on PND 13, and were therefore considered not to be adverse.


 


Based on the available results form this OECD 422 study, the NOAEL for reproduction was concluded to be at least 25 mg/kg/day. For developmental toxicity, a ANOAEL of 10 mg/kg/day (based on lower pup body weights at 25 mg/kg/day) was concluded.