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Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From: 11 Oct 2019 To: 7 May 2020
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 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
June 2018
Deviations:
no
Remarks:
Study performed in accordance with guidance and ECHA decision CCH-D-2114460631-54-01/F
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS
Justification of the study design was provided in the ECHA decision CCH-D-2114460631-54-01/F
- Premating exposure duration for parental (P0) animals: 2 weeks before mating. In the ECHA decision, ECHA noted that ten week premating exposure duration is required in case there is no substance specific information in the dossier supporting shorter premating exposure duration as advised in the ECHA Guidance on information requirements and chemical safety assessment, Chapter R.7a. In this specific case, animals of Cohort 1B are mated to produce the F2 generation. Since the premating exposure duration will be 10 weeks for these Cohort 1B animals, the fertility parameters will be covered, allowing an evaluation of the full spectrum of effects on fertility in these animals. However, the premating period shall not be shorter than two weeks and must be sufficiently long to reach a steady-state in reproductive organs as advised in the ECHA Guidance.
- Basis for dose level selection: based on two previously performed studies, which are 1) a 2-week dose-range finding study (Study No. 36510 TSR (Davies, 2010)), and 2) a reproduction/developmental toxicity screening test (OECD 421, adopted 27 Jul 1995) (Study No. 36511 RSR (Davies, 2010)) (for details see section "Details on study design").
- Inclusion of extension of Cohort 1B: in the ECHA decision, ECHA concluded that Cohort 1B must be extended to include mating of the animals and production of the F2 generation because the uses of the registered substance are leading to significant exposure of professionals and consumers and there are indications of modes of action related to endocrine disruption from available studies for the registered substance.
- Termination time for F2: no justification given.
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B: no triggers were identified for the inclusion of these cohorts (see ECHA decision).
- Exclusion of developmental immunotoxicity Cohort 3: no triggers were identified for the inclusion of this cohort (see ECHA decision).
- Route of administration: the oral route was selected as it is the appropriate route of administration for substances except gases to focus on the detection of hazardous properties on reproduction as indicated in ECHA Guidance on information requirements and chemical safety assessment (version 6.0, July 2017) Chapter R.7a. Since the substance to be tested is a solid, ECHA concluded that testing should be performed by the oral route.
- Species: the rat was chosen because it is a rodent species accepted by Regulatory Authorities for this type of study. The Sprague-Dawley strain was selected since background data from previous studies are available at Charles River Laboratories Evreux.

Test material

Constituent 1
Reference substance name:
Reference substance 001
Cas Number:
102-06-7
Test material form:
solid
Specific details on test material used for the study:
- Supplier: Sponsor
- Storage condition of test material: At room temperature; protected from humidity
- Specific handling conditions:
• Dust forming: provide appropriate exhaust ventilation at machinery and at places where dust can be generated; provide water supplies, ocular fountains and showers near the point of use
• Hygiene measures: avoid contact with skin, eyes and clothing; avoid breathing dust; wash hands after handling

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
RjHan: SD (CD®)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males approximately 11 weeks old; females approximately 10 weeks old
- Weight at study initiation: Males: mean 480 g (range: 438 g to 511 g); females mean 266 g (range: 235 g to 300 g).
- Fasting period before study: no
- Housing: The P and Cohort 1B animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust. Cohorts 1A animals were group housed. They were housed in polycarbonate cages with stainless steel lids (in 2000P cages: 4/sex per cage) containing autoclaved sawdust. Individual housing was chosen since it is preferable for pregnant animals, littering and lactating females in order to not jeopardize gestation, littering and lactation phases, and to avoid aggressive behavior around mating in males. Toward the end of gestation and during lactation, autoclaved wood shavings were provided to females and their litter as nesting material. Each cage contained objects (rat hut and nylabone) for the environmental enrichment of the animals.
- Use of restrainers for preventing ingestion (if dermal): no
- Diet: SSNIFF rat/mouse pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Tap water (filtered with a 0.22 µm filter), ad libitum
- Contaminant Analyses: The batches of diet, sawdust and wood shavings were analyzed by the suppliers for composition and contaminant levels. Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants were present in the diet, drinking water, sawdust or wood shavings at levels which could be expected to interfere with or prejudice the outcome of the study.
- Acclimation period: 8 days before treatment
- Allocation to group: during the acclimation period, the required number of animals (96 males and 96 females) was selected according to body weight and clinical condition and allocated to the groups (by sex), according to a computerized stratification procedure, so that the average body weight of each group was similar (i.e. ± 20% of the global mean values among the groups).

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C (set )
- Humidity: 50 ± 20% (set)
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h
IN-LIFE DATES: From: 7 Nov 2019 To: 7 May 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose in drinking water treated by reverse osmosis
Details on exposure:
FORMULATION PROCEDURE
- Type of test item formulation (visual observation): Suspended in the vehicle
- Preparation procedure: According to Study No. 36513 AHS (Thierry Andre, 2010) (homogeneity testing) describing the preparation procedure for a range of concentrations covering the lowest and highest used in this study.
- Frequency of preparation: According to Study No. 36513 AHS (Thierry Andre, 2010), amended in 2019.
- Storage conditions (control and test item dose formulations): At room temperature; Protected from light.
- Delivery conditions (control and test item dose formulations): At room temperature; Protected from light.

ADMINISTRATION
P and F1 generation
- Dose volume: 5 mL/kg
- Test item concentrations in vehicle: 1, 3, and 5 mg/mL, for the low, mid and high dose group respectively
- Administration: The dose formulations were administered using a plastic syringe fitted with a plastic gavage tube (in the F1 generation, juvenile gavage tubes from Days 22 to 33 p.p. and adult gavage tubes from Day 34 p.p. onwards), once a day, at approximately the same time. The quantity of the dose formulation administered to each animal was adjusted according to the most recently recorded body weight. The dose formulations were maintained under delivery conditions (at room temperature, protected from light) throughout the administration procedure. The control and test item dose formulations were stirred for 15 minutes before administration. The formulations were maintained under continuous magnetic stirring throughout the administration procedure.
Details on mating procedure:
P generation:
For scheduling purposes (to avoid excessive numbers of animals at necropsy), the mating period started over 4 different days (1st day: first six surviving males/females, 2nd day: following up to six surviving males/females and unmated pairs, 3rd day: following up to six surviving males/females and unmated pairs, 4th day: last following surviving males/females and unmated pairs).

P generation and F1 generation Cohort 1:
- M/F ratio per cage: 1/1
- Length of cohabitation: until mating, with a maximum of 14 consecutive days. The pre-coital time was calculated for each female.
- Proof of pregnancy: vaginal plug or for sperm in a vaginal lavage. The day of confirmed mating was designated Day 0 p.c.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually
- Parturition: Females were allowed to litter normally and rear their progeny until weaning (P generation), or until Day 4 p.p. (F1 generation). Any sign of a difficult or prolonged parturition (dystocia) was recorded. The morning when parturition is completed was designated Day 1 p.p. The length of gestation was calculated. Any abnormalities in nesting behavior or nursing performance were recorded.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Analytical technique: HPLC/UV method
- Principle and validation of the method: Analytical method developed and validated at Charles River Laboratories Evreux (Study No. 36512 VAA (Thierry Andre, 2010) amended in 2019) prior to dose formulation analysis. Checked parameters, acceptance criteria and obtained results are detailed in the validation report.
- Determination of test item concentrations in dose formulations: 9 dosages: two in P-premating period, and once in each P-mating, P gestation, P-lactation, F1-postweaning, F1 premating, F1 gestation and F1-lactation periods. A sample was taken from control and test item dose formulations and analyzed using the validated method
- Acceptance criterion: Measured concentration = nominal concentration ± 15%.
Duration of treatment / exposure:
P ANIMALS
- P males (at least 10 weeks of treatment): 2 weeks before mating, during the mating period (up to 14 days), until euthanasia (after euthanasia of the P females),
- P females (at least 8 to 10 weeks of treatment): 2 weeks before mating, during the mating period (up to 14 days), during gestation, during lactation until Day 21 p.p. (or Day 22 p.p. after hematology, blood chemistry and urinalysis on Day 23 p.p. for the selected females), until euthanasia for females with no evidence of mating or no delivery (25 to 26 days after the last day of the mating period).
Day 1 corresponds to the first day of the treatment period.
Day 0 p.c. corresponds to the day of confirmed mating.
Day 0 p.p. corresponds to the day on which parturition occurs.
Day 1 p.p. corresponds to the day on which parturition is completed.

F1 GENERATON
- Cohort 1A, both males and females: from weaning (Day 22 p.p.) until euthanasia (from Day 90 p.p., to Day 93 p.p. maximum).
- Cohort 1B: In the males: from weaning (Day 22 p.p.) for at least 10 weeks before mating, during the mating period (up to 2 weeks), and after euthanasia of F2 pups (on Day 4 p.p.). In the females: from weaning (Day 22 p.p.) for at least 10 weeks before mating, during the mating period (up to 2 weeks), during gestation, during lactation until Day 4 p.p. inclusive until euthanasia for females with no delivery (26 days after the last day of the mating period).
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
Low dose group
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
Mid dose group
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Remarks:
High dose group
No. of animals per sex per dose:
P: 24 per sex per dose
F1: 20 per sex per dose (Cohorts 1A and 1B)
Control animals:
yes, concurrent vehicle
Details on study design:
DOSE SELECTION RATIONALE
The dose levels were selected by the Sponsor, on the basis of the results of the following previous studies:
a) a 2-week dose-range finding study (Study No. 36510 TSR (Davies, 2010)) in which 1,3-diphenylguanidine was administered by gavage to SD rats at 0 (0.5% aqueous methylcellulose), 30, 60 and 75 mg/kg bw/day (three rats/sex/group):
• the dose levels of 60 and 75 mg/kg bw/day both exceeded the maximum tolerated dose with 50% and 17% survival rates, respectively. Prior to death or premature sacrifice, animals had general and neurological clinical signs which included lateral recumbency, clonic convulsions, staggering gait, loss of balance, locomotory difficulties, hypoactivity, mydriasis, half-closed eyes and piloerection,
• the dose level of 30 mg/kg bw/day was well tolerated by the males, the only effect being one male with loss of balance on day 9. The females, however, did not tolerate the test item well, with very low body weight gains or body weight loss and reduced food consumption. All animals survived until scheduled sacrifice and had no macroscopic abnormalities and no effects on liver, kidney, heart, ovary or testis weight,
• under the experimental conditions of this study, it was considered that the high-dose level for an OECD 421 study should be below 30 mg/kg bw/day since this dose level had an effect on the general condition of the female rats.
b) a Reproduction/developmental toxicity screening test (OECD 421, adopted 27 Jul 1995) (Study No. 36511 RSR (Davies, 2010)) in which 1,3-diphenylguanidine was administered by gavage to SD rats at 0 (0.5% aqueous methylcellulose), 5, 15 and 25 mg/kg bw/day. The dose formulations were administered daily according to the following schedule:
• in the males [4 weeks before pairing, during the pairing period (2 weeks) and until sacrifice of the females (at least 10 weeks in total)],
• in the females [4 weeks before pairing, during the mating period (2 weeks), during gestation and during lactation until day 4 post-partum inclusive (or until sacrifice) or until sacrifice for un-mated and non-pregnant females],
Treatment with the test item at 25 mg/kg bw/day resulted in reduced mean male body weight gain over the treatment period, slightly reduced mean female body weight gain during the gestation period and lower mean female food consumption during gestation and lactation. One male and one female had lateral decubitus, mydriasis and abnormal locomotion on one occasion. Mean pup body weights on day 5 of lactation and body weight gains over the lactation period were lower, although there were no treatment-related clinical signs and pup mortality was comparable with that of the controls,
There were no systemic signs of adverse toxicity at 5 or 15 mg/kg bw/day on parental and pup animals. Salivation was observed in several animals treated at 15 mg/kg bw/day and most of the animals treated at 25 mg/kg bw/day, but this was considered non-adverse.
There were no effects on mating, fertility, gestation or delivery at any dose level. There were no effects on sperm parameters, no effects on organ weights and no treatment related macroscopic or microscopic findings.
Based on the results of this study:
• the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 15 mg/kg bw/day, based on the lower body weight gain in males and females treated at 25 mg/kg bw/day,
• the No. Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 25 mg/kg bw/day,
• the NOEL for effects on the progeny was considered to be 15 mg/kg bw/day, based on the reduced body weight gain of the pups at 25 mg/kg bw/day.
Therefore, as 30 mg/kg bw/day proved to alter the general condition of the female rats in the 2 week dose range finding study (Study No. 36510 TSR (Davies, 2010)), the dose levels used for the OECD 421 study (Study No. 36511 RSR (Davies, 2010)) were retested in the present study.

CONSTITUTION OF THE F1 GENERATION
On Day 22 p.p., pups from all available litters (up to 20 litters per group) were assigned to two Cohorts of animals, as follows:
• Cohorts 1A = Reproductive/developmental toxicity testing,
• Cohorts 1B = Reproductive/developmental toxicity testing with extension to produce an F2 generation.
Pups were selected randomly, with the exception that obvious runts (animals with a body weight more than two standard deviations below the mean pup weight of the respective litter) were not included, as they are unlikely to be representative of the treatment group.
The pups selected to constitute the F1 Cohorts were identified by an implanted microchip (unique Charles River Laboratories Evreux identity number) at weaning. Day 22 p.p. was designated Day 1 of the F1 generation.

F1 GENERATION COHORTS
Cohort 1A: On Day 22 p.p., one male and one female/litter/group (20/sex/group) was selected for assessment of effects upon reproductive systems and of general toxicity. Whenever necessary, partial adjustment (for example 2 males or 2 females/litter) was permitted.
The estrous cycle stage was determined for all females in Cohort 1A from a fresh vaginal lavage (stained with methylene blue), each morning (between 07:30 and 10:00 a.m.) as follows:
• after the onset of vaginal patency, until the first cornified smear was recorded (estrous), in order to determine the time interval between these two events,
• at least a period of 2 weeks towards the end of the treatment period (same calendar dates for all females).
Cohort 1B: On Day 22 p.p., one male and one female/litter/group (20/sex/group) was selected for follow-up assessment of reproductive performance by mating F1 animals and to obtain additional histopathology data (1). Whenever necessary, partial adjustment (for example 2 males or 2 females/litter) was permitted.
Males and females of the same dose group were cohabited (avoiding the pairing of siblings) for up to 2 weeks or until mating, beginning on Days 91-96 p.p.

F2 GENERATION
The F2 litters were terminated on Day 4 p.p.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
MORBIDITY AND MORTALITY (P and F1 generation): Yes
Time schedule: each animal once a day during the acclimation period (P generation) and at least twice a day during the treatment period, including weekends and public holidays (P and F1 generation).

CLINICAL SIGNS (P and F1 generation): Yes
Time schedule: each animal once a day as part as routine examinations, from arrival (P generation). From the start of the treatment period, each animal was observed at least twice a day (before and after treatment), at approximately the same time of day, for the recording of clinical signs (P and F1 generation).

DETAILED CLINICAL OBSERVATIONS (P and F1 generation): Yes
Time schedule: all animals once a week until the end of the study (P and F1 generation).
Observations (P and F1 generation): included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards).

BODY WEIGHT (P and F1 generation): Yes
Time schedule: each male was weighed once during the acclimation period (P generation), on the first day of treatment (Study Day 1), then at least once a week until euthanasia (P and F1 generation).
Each female was weighed once during the acclimation period (P generation), on the first day of treatment (Day 1), then once a week (P generation and Cohorts 1A and 1B) until mated (or until euthanasia for females with no evidence of mating), on Days 0, 4, 7, 10, 14, 17 and 20 post-coitum (p.c.) (P generation and Cohort 1B) and, on Days 1, 4, 7, 14 and 21 p.p. (P generation) or on Days 1 and 4 p.p. (Cohort 1B).
All animals were weighed at euthanasia as normal procedure before pathology examination.

FOOD CONSUMPTION AND COMPOUND INTAKE (P and F1 generation): Yes
Time schedule: the quantity of food consumed by each male was measured once a week from the first day of treatment (P generation and Cohorts 1A and 1B) until the start of the mating period and then after the mating period until euthanasia (P generation and Cohort 1B).
The quantity of food consumed by each female was measured once a week from the first day of treatment until euthanasia (Chort 1A) or until the start of the mating period, during gestation for the intervals: Days 0-4, 4-7, 7-10, 10-14, 14-17 and 17-20 p.c. (P generation and Cohort 1B) and during lactation for the intervals: Days 1-4, 4-7, 7-14, and 14 21 p.p. (P generation) or for the interval: Days 1-4 p.p. (Cohort 1B).
During the mating period, food consumption was not measured for males or females.

WATER CONSUMPTION: No

HEMATOLOGY AND COAGULATION (P generation and Cohort 1A): Yes
Blood samples were taken from the orbital sinus of 10 male and 10 female animals (under light isoflurane anesthesia) into tubes containing the appropriate anticoagulant.
Time point: Day of necropsy
Fasting: yes, overnight period of at least 14 hours
Parameters analysed: Red blood cell count, Mean cell volume, Packed cell volume (hematocrit), Hemoglobin, Mean cell haemoglobin concentration, Mean cell haemoglobin, Trombocyte (platelet) count, Total leucocyte count, Reticulocytes and Differential white cell count with cell morphology (Neutrophils, Eosinophils, Basophils, Lymphocytes, Large unstained cells, Monocytes), prothrombin time (blood clotting time), Fibrinogen, and Activated partial thromboplastin time.

CLINICAL CHEMISTRY (P generation and Cohort 1A): Yes
Blood samples were taken from the orbital sinus of 10 male and 10 female animals (under light isoflurane anesthesia) into tubes containing the appropriate anticoagulant.
Time point: Day of necropsy
Fasting: yes, overnight period of at least 14 hours
Parameters analysed: Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Glucose, Urea, Creatinine, Total bilirubin, Total cholesterol, Triglycerides, Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Total proteins, Albumin, Albumin/globulin ratio, and Bile acids.

BLOOD SAMPLES FOR FURTHER POSSIBLE ANALYSES (P generation and Cohort 1A): Yes
Blood samples for further possible analyses were taken at termination (approximately 1 mL per animal) into a plain tube.

URINALYSIS (P generation and Cohort 1A): Yes
For urine collection, the animals were individually placed in metabolism cages an overnight period of at least 14 hours. The urine was collected onto thymol crystals.
Time point: Day of necropsy
Fasting: yes, overnight period of at least 14 hours
Parameters analysed: Appearance, Color, Volume, pH, Specific gravity, Proteins, Glucose, Ketones, Bilirubin, Nitrites, Blood (hemoglobin), Urobilinogen (non-GLP), and Cytology of sediment (Leucocytes, Erythrocytes, Cylinders, Magnesium ammonium phosphate crystals, Calcium phosphate crystals, Calcium oxalate crystals, Epithelial cells).

THYROID HORMONE ANALYSIS (P generation and Cohort 1A): Yes
Blood samples (approximately 0.5 mL) were taken (between 07:30 and 10:00 a.m.) from the orbital sinus of 10 P male and 10 lactating P female animals and the first 10/sex/group Cohort 1A animals under isoflurane anesthesia into tubes containing K3-EDTA as anticoagulant.
Time point: Day of necropsy
Fasting: No, except for animals also sampled for hematology, blood biochemistry and urinalysis.
Parameters analysed: thyroid hormone (T4) and thyroid stimulating hormone (TSH) levels.
Oestrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning (between 07:30 and 10:00 a.m.) as follows:
• during the 2 weeks of the premating period (P generation),
• during the mating period, until the females were mated, or the mating period had ended (P and F1 generation Cohort B).
Sperm parameters (parental animals):
Full seminology investigations in P and Cohort 1A male parental generations from the control and high-dose groups (groups 1 and 4) included the determination of: testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, and sperm morphology.
Sperm motility and morphology investigations of both the testis and epididymis were also performed on all surviving P and Cohort 1A males from the low- and intermediate-dose groups (groups 2 and 3).
Litter observations:
LITTER SIZE (F1 and F2 pups)
- Recorded on Day 1 p.p.
- Observation: daily in order to note the number of live, dead and cannibalized pups.

STANDARDISATION OF LITTERS (F1 pups)
- Performed on day 4 postpartum: Yes
- To obtain as nearly as possible 5 males and 5 females per litter; partial adjustment (for example 6 males and 4 females) was permitted; excess pups were killed and discarded.

CLINICAL SIGNS (F1 and F2 pups): Yes
- Time schedule: each pup daily
- Daily observations: clinical signs, abnormal behavior and external abnormalities (including oral cavity and orifices).
- Observation on the first clinical examination (on Day 1 p.p.): gross external examination (e.g. external visible abnormalities, cleft palate, subcutaneous hemorrhages, abnormal skin color or texture; presence of umbilical cord, lack of milk in the stomach, presence of dried secretion), qualitative assessment of body temperature, activity and reaction to handling

BODY WEIGHT (F1 and F2 pups): Yes
- Time schedule: each live pup on Days 1, 4, 7, 14 and 21 p.p (F1 pups) and on Days 1 and 4 p.p. (F2 pups).

PARAMETERS EXAMINED (F1 pups):
- Anogenital distance (AGD) on Day 1 p.p. The AGD was normalized to the cube root of body weight recorded on Day 1 p.p.
- Number of nipples and of areolae in male pups: on Day 12 p.p.

SEXUAL DEVELOPMENT (F1 pups): Yes
All male animals were observed each day from Day 35 p.p. (i.e. Day 14 of F1 generation), until cleavage of the balanopreputial groove (preputial separation) was observed (until euthanasia for Cohorts 1A animals, or until mating for Cohort 1B).
All female animals were observed each day from Day 25 p.p. (i.e. Day 4 of F1 generation), until vaginal opening was observed (until euthanasia for Cohorts 1A, or mating for Cohort 1B).
The body weight of each animal was recorded on the day of the positive measurement.

THYROID HORMONE ANALYSIS (F1 Culled Pups, and F2 Pups): Yes
Blood samples were taken (between 07:30 and 10:00 a.m.) into tubes containing K3-EDTA as anticoagulant.
• on Day 4 p.p. from F1 culled pups and from F2 pups euthanized (as much blood as possible was collected by decapitation under isoflurane anesthesia and then pooled per litter), for potential measurements of thyroid hormone (T4) levels,
• on Day 22 p.p. from F1 pups not selected for Cohorts (at least 0.25 mL per pup was collected from the vena cava immediately after euthanasia), into individual tubes for potential measurements of thyroid hormone (T4) and thyroid stimulating hormone (TSH) levels,
Fasting: No

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
At scheduled euthanasia, adult animals of P generation and Cohort 1A were deprived of food for an overnight period of at least 14 hours.
On completion of the treatment period, all surviving P animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination: P males after weaning of the F1 progeny, P females on Days 23 p.p., P females which did not deliver: on Days 25-26 p.c., P female with no evidence of mating: 25 days after the end of the mating period, and P females with total litter loss: as appropriate.
On completion of the treatment period, all surviving F1 animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination: F1 males (Cohort 1A): on Days 90-92 p.p., F1 males (Cohort 1B): after euthanasia of the F2 progeny, F1 females (Cohort 1A): on Days 91-93 p.p., F1 females (Cohort 1B): on Day 4 p.p., F1 females which did not deliver: on Day 25 p.c., and F1 females with total litter loss.
Prematurely ethanisation was performed by an intraperitoneal injection of sodium pentobarbital followed by exsanguination of females during the mating or lactation period. Prematurely ethanisation was performed by inhalation of carbon dioxide gas followed by cervical dislocation of females during the gestation period and of females with difficulties of delivery.

GROSS NECROPSY
All animals found dead or prematurely euthanized were submitted for a complete macroscopic post mortem examination. Macroscopic lesions were preserved in appropriate fixative. In all females prematurely euthanized, the brains were preserved in appropriate fixative. Of females euthanized during the mating, gestation periods, during delivery, or lactation period the following parameters were determined where applicable: pregnancy status, the numbers of corpora lutea, implantation sites and classified as live or dead concepti, early or late resorptions or scars.

A complete macroscopic post-mortem examination was performed on all P and F1 animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.
The numbers of implantation sites were recorded for females euthanized on Day 23 p.p. (P generation) and on Day 4 p.p. (Cohort 1B).The numbers of corpora lutea and implantation sites were recorded for female (P generation) euthanized 25 days after the end of the mating period with no evidence of mating and for females euthanized on Days 25-26 p.c. due to no delivery.

HISTOPATHOLOGY / ORGAN WEIGHTS
The body weight of each animal euthanized as scheduled was recorded before euthanasia. For these animals, the organs (according to Guideline) were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated. Tissue collection and preservation were according to guideline.
In view of the neurological clinical signs, the brains from females of the P generations were trimmed as recommended by Bolon et al. (2013) for neurotoxicity studies, with dedicated seven brain sections.

LYMPHOCYTE SUBTYPING (Cohort 1A)
Half of the spleen of 10 males and 10 females per group from Cohort 1A animals were subject to a splenic lymphocyte subpopulation analysis (T lymphocytes, CD4+ and CD8+ T lymphocytes, B lymphocytes, Natural Killer (NK) cells and NKT cells). Lymphocyte subtyping was carried out by flow-cytometry.
Postmortem examinations (offspring):
SACRIFICE
F1 and F2 pups were euthanized by an intraperitoneal injection or sodium pentobarbital followed by exsanguination: moribund pups (F1 and F2), pups whose mother died as soon as possible (F1 and F2), pups not selected on Day 4 p.p. (F1), pups on Day 4 p.p. (F2), and pups not selected at weaning on Day 22 p.p. (F1).

GROSS NECROPSY
A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all pups found dead (except cannibalized) and prematurely euthanized pups. Special attention was paid to the reproductive organs and to whether the pup has fed (e.g. presence of milk in the stomach). Macroscopic lesions were preserved in appropriate fixative.
A complete macroscopic post-mortem examination was performed on all F1 pups culled on Day 4 p.p. and not selected F1 pups on Day 22 p.p., and on F2 pups, and prematurely euthanized or found dead pup. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
The body weight of each animal euthanized as scheduled was recorded before euthanasia. For these animals, the organs (according to Guideline) were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated. Tissue collection and preservation were according to guideline.
Statistics:
Body Weight, Food Consumption and Reproductive data were compared by one-way analysis of variance and Dunnett's test, (mean values being considered as normally distributed, variances being considered as homogeneous) or by Fisher’s exact probability test (proportions).
Statistical analysis of organ weight data (level of significance of 0.05 or 0.01) was performed according to a decision logic as indicated in sections 6.15.2 in the study report.
Statistical analysis according to a decision logic as indicated in sections 6.15.3 in the study report was used for: Number of Primary Follicles/Corpora Lutea, Ano-Genital Distance, Number of Nipples and Areolae, Time of Preputial Separation/Vaginal Opening, Time to First Estrous after Vaginal Opening/Patency, Seminology, Hematology, Blood Biochemistry, Urinalysis, Thyroid Hormones, Post-Implantation Loss, Sex Ratio, Live Birth, Viability and Lactation Indexes
Statistical analysis of Splenic Lymphocyte Immunophenotyping was performed according to a decision logic as indicated in sections 6.15.4 in the study report.
Reproductive indices:
The following parameters were calculated:
post-implantation loss:
(Number of implantation sites - Number of live pups / Number of implantation sites) x 100
mating index:
(Number of mated animals / Number of paired animals) x 100
fertility index:
(Number of pregnant female partners / Number of mated pairs) x 100
gestation index:
(Number of females with live born pups / Number of pregnant females) x 100
Offspring viability indices:
live birth index:
(Number of live pups on Day 1 p.p. / Number of delivered pups) x 100
viability index on Day 4 p.p.:
(Number of surviving pups on Day 4 p.p. (before culling) / Number of delivered pups) x 100
lactation index:
(Number of surviving pups on Day 21 p.p. / Number of surviving pups on Day 4 p.p. (after culling)) x 100
AGD/cube root of body weight ratio (calculated with Excel):
AGD / ³vBody weight

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Ptyalism was observed in all groups including controls, but the incidence was higher in test item treated groups. This finding (associated with reflux at dosing and/or soiled mouth at 25 mg/kg bw/day) is commonly observed after a gavage procedure and was considered to be test item treatment-related but not adverse.

Ptyalism was observed in all treated groups. This finding is commonly observed after a gavage procedure and was considered to be test item treatment-related but not adverse. At 25 mg/kg bw/day and at the end of the pregnancy period (from Study Day 42), there was a series of clinical signs suggestive of neurologic disorders in 8 out of 18 surviving females (e.g. clonic convulsion, locomotory difficulties, loss of balance, staggering gait and/or tonic seizures). These findings were transient and observed after dosing only (mainly not observed on next days after appearance). These findings were considered to be test-item related and adverse. Other findings observed with a low and/or similar incidence across groups (including controls) were those that are commonly observed in this species/strain of animals or in pregnant animals, and they were therefore not considered to be test item-related.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
In the low dose group, 2 females were prematurely euthanized. One female was euthanized on Day 1 p.p. because of the dead litter. This female showed piloerection, hunched posture pallor of eyes/extremities and reddish vaginal discharge before sacrifice. The clinical signs and dead litter were considered to be test-item related. The other female animal was euthanized on Day 2 p.p. (ulcerated mass). This death was not considered to be test-item related.
In the mid dose group, 2 females were prematurely euthanized. One female was euthanized on Day 2 p.p. because of the dead litter. This female showed showed no clinical sign before sacrifice. This death was considered to be test-item related. The other female animal was euthanized on Day 24 p.c. because of the difficulties to deliver associated with an abdomen increased in size. This female showed severe clinical signs (piloerection, hunched posture, generalized pallor, vaginal discharge) before sacrifice. All findings were considered to represent evidence of dystocia and to be test-item related.
In the high dose group, 6 females were prematurely euthanized. Two females were euthanized on Day 1 p.p. because of cold to the touch, lateral recumbency, hypotonia, tonic seizures, tonic convulsion and/or dyspnea. Three females were euthanized on Day 1 p.p. because of dead litter. The females showed piloerection, hunched posture, pallor of eyes/extremities, ventral recumbency, staggering gait, exophthalmos and/or dyspnea before sacrifice. One female was euthanized on Day 23 p.c. because of the difficulties to deliver. The female showed cold to the touch, lateral recumbency, tonic seizures, clonic convulsion, dyspnea and exophthalmos before sacrifice. All deaths in the high dose group were considered to be test-item related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the pre- (males and females) and post-mating (males) period of the P generation, there were few statistically significant differences on the mean body weight or mean body weight change when compared with controls. However, there were no dose-related effects on mean body weight or mean body weight change in males or females. Therefore, any relationship to a test item-treatment was considered to be unlikely.
During the pregnancy period, there was a single statistically significant difference when compared to controls in the mid dose group. This was incidental and not following a dose response relationship. Therefore, any relationship to a test item-treatment was considered to be unlikely.
During the lactation period, mean body weight changes was increased on Days 7 to 14 p.p. (+21g vs. +7 g in controls, p<0.01) resulted in higher mean body weight on Days 14 and 21 p.p. (up to + 5% vs. controls on Day 14 p.p, p<0.05) in the high dose group. A test item-relationship cannot be ruled out but taking into account the amplitude of the changes, these findings were considered to be non-adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During the pre- (males and females) and post-mating (males) period of the P generation, there were few statistically significant differences on the mean food consumption when compared with controls. However, there were no dose-related effects on mean food consumption in males or females. Therefore, any relationship to a test item-treatment was considered to be unlikely.
During the pregnancy period, mean food consumption was lower between Days 14 and 20 p.c. (down to -11% on Days 17 to 20 p.c., p<0.001) in the high dose group. This finding was considered to be test-item related and non-adverse taking into account the amplitude of the changes.
During the lactation period, mean food consumption was lower from Day 1p.p. with statistically significance on Days 4 to 7 p.p. (-14%, p<0.05) in the high dose group. While a test item-relationship could not be excluded, this finding was considered to be non-adverse.
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):
In the females, there was a significant (P<0.05) higher number of mean cell hemoglobin in the mid and high dose group (19.0 in both groups) when compared with the control group (18.3). There were no test item treatment-related effects on hematology or coagulation in P generation animals. In females, the statistically significant differences were of minimal amplitude and were not observed in males. In addition, all values remained within the range of Historical Control Data (17.0-21.0). Therefore, a test item relationship was considered to be unlikely.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
When compared with the control group, there were a couple of statistically significant differences in P generation males. The statistically significant differences were recorded with no dose relationship, were not observed in females and remained within the range of Historical Control Data. Therefore, a test item relationship was considered to be unlikely.
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
THYROID HORMONES
In P generation males and when compared with controls, there were no statistically significant differences on mean TSH and T4 plasma levels. In P generation females and at 25 mg/kg bw/day, when compared with controls or Historical Control Data, there were high mean T4 concentration (+38%, p<0.05) associated with low mean TSH concentrations (-14%). A test item-relationship cannot be excluded but in the absence of associated macroscopic or microscopic findings, these differences were considered not to be adverse. There were no effects at 15 and 5 mg/kg bw/day.
Urinalysis findings:
no effects observed
Description (incidence and severity):
When compared with controls, there were no statistically significant differences on urinalysis parameters in P generation animals. In males of the high dose group, the apparent increased urinary volume when compared with controls was mainly due to one animal. Therefore, any test item relationship was considered to be unlikely.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were no test item-related findings in the brain with dedicated examination.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related changes were noted in the liver and consisted in non-adverse dose-related minimal hepatocellular hypertrophy in males (7 out of 24 animals, grade 1) and females (1 out of 22 animals, grade 1) treated at 25 mg/kg bw/day. The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.
There were test item-related changes neither in the male reproductive organs, including the detailed examination of the testes using a thorough understanding of tubule development through the different stages of the spermatogenic cycle, or in the female reproductive organs.
There was a good correspondence between the vaginal smears and the histopathological examination of estrus cycle, except in one control female (histologically in diestrus while the smear demonstrated intermediate and superficial cells with low cellularity).

At enumeration of corpora lutea in the ovary of high-dose group and control group, there were no statistical differences with a mean number of corpora lutea of 12.11 (±3.22) and 10.04 (±3.58) per animal respectively. At enumeration of the number of primordial follicles, there were no statistical differences between the high-dose and control groups, with a mean number of 6.51 (±4.83) and 5.91 (±3.92) primordial follicles per animal on PCNA-stained slides, respectively.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
A summary of main microscopic lesions at necropsy in parental cohort are presented in Table 18 of the section "Any other information on results incl. tables". One female, died at Day 40, had a mammary gland adenocarcinoma. Spontaneous mammary gland adenocarcinoma can be seen in untreated females rats of this age (Kuzutani et al., 2012) and therefore was considered not treatment related.
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
In the P-generation at 25 mg/kg bw/day and when compared with controls, mean number of days of estrus was high (4.3 vs. 3.5 days, p<0.01) and mean number of days of metestrus was low (3.3 vs. 4.9 days, p<0.001). A test item-relationship cannot be excluded. However, in the absence of any effect on mean number of cycles, mean duration of cycles, mean percent of females cycling normally or mean percent of females with all stages, this finding was considered to be non-adverse.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
In test item-treated groups and when compared with controls, there were no effects (no statistically significant differences) on sperm parameters (motility, morphology, sperm/testicular numerations or daily sperm production rate).
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
MATING AND FERTILITY DATA
There were no effects on mating (including the mean number of days taken to mate) or fertility in the P generation.

DELIVERY DATA
The summary of delivery data and distribution of gestation periods is presented in Table 3 and Table 4, respectively, of the section "Any other information on results incl. tables". When compared with controls, there were no statistically significant differences in mean duration of gestation. However, there was a trend of increased gestation period on an individual values basis. Two females at 5 mg/kg bw/day had 24-day gestation periods associated with a high pup mortality rate [one female with 1 live pup and 9 dead pups, and another female with a dead litter (14 pups)]. At 25 mg/kg bw/day, there was an increased number of females with 23-day gestation periods when compared to the control group, which was associated to a high post-implantation loss.
From 5 mg/kg bw/day, when compared with controls (despite the absence of statistical significance) and Historical Control Data, there was a dose-related tendency towards lower number of viable pups (down to 9.3 vs. 12.0 live pups on Day 1 p.p. at 25 mg/kg bw/day) and live birth index (down to 72.8 vs. 95.1% at 25 mg/kg bw/day). At 25 mg/kg bw/day, there was a high mean percentage of post-implantation losses (27.1 vs. 15.6%, p<0.05) while no changes were observed at 5 and 15 mg/kg bw/day.
Therefore, a test-item relationship between these increased gestation periods, the high mean percentage of post-implantation losses and deaths in pups (i.e. low live birth index from 5 mg/kg bw/day) cannot be excluded. These findings were considered to be adverse.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
Remarks on result:
other: Without test item-related findings in the brain
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive/ developmental toxicity
Effect level:
< 5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Remarks on result:
other: Was not associated with maternal neurotoxicity which was only observed in the high dose group

Target system / organ toxicity (P0)

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
25 mg/kg bw/day (actual dose received)
System:
nervous system
Organ:
brain
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
5 mg/kg bw/day (actual dose received)
System:
female reproductive system
Organ:
uterus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A
Ptyalism was observed in all test item-treated groups (males and females) and with increased incidences. This finding is commonly observed after gavage and was considered to be test item treatment-related but not adverse.
Other findings observed with a low and/or similar incidence across groups (including controls) are those that are commonly observed in this species/strain of animals or in pregnant animals, and they were therefore not considered to be test item treatment-related.

Cohort 1B
At 25 mg/kg bw/day and in both sexes, there was a series of clinical signs suggestive of neurologic disorders (e.g. clonic/tonic convulsion, loss of balance and/or staggering gait) which were observed mainly from Study Day 94 after dosing for 1 to 3 days. Taking into account the nature and incidences of these finding, these observations were considered to be test-item-related and adverse.
Ptyalism was observed with an increased incidence in test item-treated groups when compared with controls. This finding is commonly observed after gavage and was therefore considered to be test item treatment-related but not adverse. Other findings observed with a low and/or similar incidence across groups (including controls) are those that are commonly observed in this species/strain of animals or in pregnant animals, and they were therefore not considered to be test item treatment-related.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Cohort 1A
There were no unscheduled deaths in Cohort 1A animals.

Cohort 1B
There were no unscheduled deaths in males. In females, there was one animal in the high dose group found dead on study day 1, without showing adverse clinical signs the day before death. Additionally, 3 animals were euthanized in the control group, 0 in the low dose group, 3 in the mid dose group and 5 in the high dose group. Two of the euthanized animals in the mid dose group and all of the euthanized animals in the high dose group were considered to be test item-related. In the mid dose group, 2 females were euthanized for dead litter on Day 1 p.p. and one female for no delivery (not pregnant). In the high dose group, 2 females were euthanized for dead litter on Day 1, one female because of lateral recumbency and loss of balance, one female because of hypoactivity, lateral recumbency and clonic convulsion, and one female because of difficulties to deliver on Day 24 p.c.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A
There were no adverse effects on mean body weight or mean body weight changes. When compared with controls, the few statistically significant differences were recorded without any dose level relationship and with minimal amplitudes. Therefore, any test item treatment-related effect was considered to be unlikely.

Cohort 1B
During the pre- (males and females) and post-mating (males) periods, there were no adverse effects on mean body weight or mean body weight change in Cohort 1B animals. The few statistically significant differences (recorded without a dose level relationship and with minimal amplitude when compared with controls) were considered to be fortuitous.
During the pregnancy period, there were no adverse effects on mean body weight or mean body weight change in Cohort 1B females. At 15 and 25 mg/kg bw/day, statistically significant increases in body weight were observed. They were of minimal amplitudes when compared with controls and considered to be fortuitous during the pregnancy period (these increased mean body changes were mainly gained during the pre-mating period).
During the lactation period, there were no adverse effects on mean body weight or mean body weight change in Cohort 1B females. The few statistically significant differences were of minimal amplitude when compared with controls and with no dose level relationship. Therefore, they were considered to be fortuitous.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A
There were no effects on mean food consumption.

Cohort 1B
During the pre- (males and females) and post-mating (males) periods, there were no test item treatment-related effects on mean food consumption in Cohort 1B animals. The few statistically significant differences were of minimal amplitude, observed without any dose level relationship and therefore considered to be fortuitous.
During the pregnancy period, no adverse effects on mean food consumption in Cohort 1B females. The statistically significant difference was isolated and of minimal amplitude. Therefore, this difference was considered to be fortuitous.
During the lactation period, there were no significant effects on mean food consumption in Cohort 1B females during the lactation period.
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):
Cohort 1A
There were no test item-related effects. A statistically significant difference was recorded in low-dose male group (LUC: 0.19 vs. 0.12 G/L in controls, p<0.01). There was no similar finding in females, no dose response and the value remained within the range of Historical Control Data. Therefore, a test item-relationship was considered to be unlikely.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A
There were no test item treatment-related effects on blood biochemistry in Cohort 1A animals. The statistically significant differences in Cohort 1A animals were recorded with no dose relationship, were not observed in both sexes and/or remained within the range of HCD. Therefore, a test item-relationship was considered to be unlikely.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A
In Cohort 1A males and when compared with controls or Historical Control Data, there were no effects on mean thyroid hormones levels (T4 and TSH). The few statistically significant differences were observed without any dose level relationship and/or remained within the range of Historical Control Data. In addition, there were no macroscopic or microscopic test item-related findings in the thyroid glands.
In Cohort 1A females and when compared with controls, there were no effects on T4 and TSH plasma levels. In addition, there were no macroscopic or microscopic test item-related findings in the thyroid glands.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A
From 15 mg/kg bw/day onwards in males and at 25 mg/kg bw/day in females, there were increases in mean urinary volume when compared with controls (+33% in males, down to p<0.01 and +27% in females, not statistically significant). Taking into account the low amplitude of the changes, this finding was considered to be non-adverse.
Behaviour (functional findings):
not examined
Immunological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A
In males and when compared with controls, there were statistically significant increase of NK cells at 15 mg/kg bw/day only) both in terms of relative (4.8 vs. 3.4%, p<0.01) and absolute counts (11397 cells/mg of spleen compared to 7001 cells/mg of spleen) counts and, in terms of relative counts, a decrease of T cells (36.6 vs. 42.5%, p<0.01).
In females and when compared with controls, there was a statistically significant decrease of NK cells at 25 mg/kg bw/day, both in terms of relative (2.6 vs. 3.8% of splenocytes, p<0.01) and absolute (5879 vs. 10713 cells/mg of spleen, p<0.01) counts.
Taken into account the low amplitudes of the changes when compared with controls and/or the absence of a dose-level relationship, a test-item effect was considered to be unlikely.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A
Relevant changes in mean final body weights and organ weights in treated groups are presented in Table 20 of the section "Any other information on results incl. tables". Increased absolute and relative-to-body liver weights were recorded in females treated at = 5 mg/kg bw/day and in relative-to-body weights in males treated at 25 mg/kg bw/day (up to +18%; p<0.01 or 0.05). This correlated with hepatocellular hypertrophy at microscopic examination.
Increased absolute and relative-to-body mesenteric lymph node weights were recorded in females treated at = 15 mg/kg bw/day (up to +30%; p<0.01 or 0.05). There were no microscopic correlates. The relationship to test item administration was thus considered to be unlikely.
The other organ weight changes were not considered to be test item related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings.

Cohort 1B
Relevant changes in mean final body weights and organ weights in treated groups are presented in Table 22 of the section "Any other information on results incl. tables". Increased absolute and/or relative-to-body liver weights were recorded in males and females treated at = 15 mg/kg bw/day.
The other organ weight changes were not considered to be test item-related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings. This included the absolute and relative-to-body kidney weights that were minimally increased in females treated at 15 mg/kg bw/day when compared to controls. In the absence of dose-relationship and of similar differences in the other cohorts, this change was considered to be unrelated to the test item administration.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A
There was no unscheduled mortality. There were no test item-related findings at macroscopic post-mortem examination. The few isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age. This included the black discoloration seen in the stomach from three high-dose males (correlated generally with background erosion), that was seen also in low- and mid-dose females.

Cohort 1B
A summary of main macroscopic lesions at necropsy in Cohort 1B are presented in Table 21 of the section "Any other information on results incl. tables". The few isolated gross observations at macroscopic post-mortem examination were considered to be consistent with spontaneous findings encountered in the rats of these strain and age. This included the thickened uterus that correlated with remnants of gestational glands in several females treated at 15 or 25 mg/kg bw/day, and the black mass located in the uterus of 2 out of 15 females treated at 25 mg/kg bw/day. This was not associated with reproduction trouble and correlated with placental remnants bearing necrosis and hemorrhage at microscopic examination.
Neuropathological findings:
no effects observed
Description (incidence and severity):
Cohort 1B
There were no test item-related findings in the brain with dedicated examination.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A
Test item-related changes were noted in the liver and consisted in non-adverse dose-related minimal hepatocellular hypertrophy in males (1 out of 20 animals, grade 1) and females (14 out of 2 animals, grade 1) treated at 25 mg/kg bw/day. The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.
There were test item-related changes neither in the male reproductive organs, including the detailed examination of the testes using a thorough understanding of tubule development through the different stages of the spermatogenic cycle, or in the female reproductive organs.
There was a good correspondence between the vaginal smears and the histopathological examination of estrus cycle in all examined females.

At enumeration of corpora lutea in the ovary of high-dose and control groups, there were no differences, with a mean number of corpora lutea of 18.45 (±6.60) and 19.00 (±7.36) per animal respectively, reaching no statistical significance. The mean number of primordial follicles was lower in females treated at = 5 mg/kg bw/day when compared to the control group (8.68±6.77 in the low dose group versus 9.31±6.09 in the control group). However, these differences did not reach statistical significance and did not correlate with any microscopic changes in the reproductive system. Thus, these differences were considered to be unrelated to the test item administration.

Cohort 1B
Slight degeneration/necrosis in placenta with hemorrhage, accumulation and/or mineralization was seen in 2 out of 20 females treated at 25 mg/kg bw/day. There was also minimal focal hemorrhage in the uterus from one female treated at 15 mg/kg bw/day. These females had no reproduction troubles. The relationship to test item was considered to be unlikely, in the absence of microscopic examination of the control group from this cohort, and of the absence of similar changes in the cohort 1A.
In the other examined organs, there were no test item-related findings, including in the brain with dedicated examination.
The few microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1B
One control female had a spontaneous histiocytic sarcoma in spleen, lungs and liver, and one high-dose female had a mammary gland adenocarcinoma. This tumor is common in the rats of these strain and age (see discussion), and thus was considered not to be related to the test item administration.
Other effects:
no effects observed
Details on results:
SEXUAL MATURATION
Cohort 1A
There were no effects on mean age at balanopreputial separation or on mean body weight on the day of occurrence in Cohort 1A males. There were no effects on mean age at vaginal opening or on mean body weight on the day of occurrence in Cohort 1A females. In the 5 mg/kg bw/day group, one female did not had a complete vaginal opening until euthanasia. This female had a fine membranous vaginal remnant (a common observation in this strain) which is known to have no impact mating success. There were no effects on mean time (number of days) to first estrous after vaginal opening in Cohort 1A females.

Cohort 1B
There were no effects on the mean age of balanopreputial separation or on mean body weight on the day of occurrence in Cohort 1B males. In the 5 and 25 mg/kg bw/day groups, two males had an incomplete preputial separation until euthanasia. This is a common finding in this species and strain for which a test item-relationship was considered to be unlikely.
There were no effects on the mean age at vaginal opening or on mean body weight on the day of occurrence in Cohort 1B females.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no effects on mean estrous cycle parameters in Cohort 1A females.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
In test item-treated groups and when compared with controls, there were no relevant effects on sperm analysis parameters (motility, morphology, sperm/testicular numerations or daily sperm production rate).
Reproductive performance:
no effects observed
Description (incidence and severity):
MATING AND FERTILITY DATA
There were no effects on mating and fertility in test item treated groups when compared with controls or Historical Control Data in the Cohort 1B animals.

DELIVERY DATA
There were no adverse effects on pups sex ratio. From 5 mg/kg bw/day, there was a dose-related increase in the mean percent of post-implantation losses (up to 24.6% at 25 mg/kg bw/day vs. 13.6% in controls) associated with low live birth index from 15 mg/kg bw/day (down to 67.5% at 25 mg/kg bw/day vs. 100.0% in controls) and low number of live pups at 25 mg/kg bw/day (7.8 vs. 11.4 in controls, p<0.0). These findings were considered to be test-item-related and adverse from 15 mg/kg bw/day.
At 25 mg/kg bw/day and when compared with control, mean duration of gestation was increased (22.3 vs. 21.9 days in controls, p<0.05). Indeed, there were 0/16, 0/20, 1/18 and 7/19 females with 23 days of gestation in control, low-, mid- and high-dose groups, respectively. This finding was considered to be test-item related and adverse.
When compared with controls, the statistically significant difference in the mean number of implantation (15.7 vs. 13.1, p<0.01) was observed at 15 mg/kg bw/day without any dose level relationship and therefore considered to be fortuitous.

Details on results (P1)

DISCUSSION ON PATHOLOGY
There was a dose-related increased incidence of mortality in test item-treated females (P generation and cohort 1B) that was considered to be related to the test item administration at = 5 mg/kg bw/day (parents) and = 15 mg/kg bw/day (cohort 1B), although not recorded in cohort 1A.
One control female (cohort 1B), four females treated at 25 mg/kg bw/day (two in P generation and two in cohort 1B) were euthanized for humane grounds, due to the severe clinical signs.
In P generation, there was reproduction trouble (dead litter or difficulty to deliver) in one female treated at 5 mg/kg bw/day, two females treated at 15 mg/kg bw/day and in four females treated at 25 mg/kg bw/day. For cohort 1B, there was a reproduction trouble in two females treated at 15 mg/kg bw/day and in three females treated at 25 mg/kg bw/day.
Conversely, the low dose female Q28908 (parental cohort P generation) and the high-dose Q29125 (cohort 1B) had a spontaneous mammary gland adenocarcinoma that can be seen in untreated females rats of this age (Kuzutani et al., 2012).
In the liver from P generation and cohort 1A animals treated at 25 mg/kg bw/day, centrilobular or diffuse hepatocellular hypertrophy was observed and correlated along with increased liver weights at = 15 or 25 mg/kg bw/day in males and/or females, with no gross correlates.
The liver hypertrophy was considered to be secondary to the induction of hepatic microsomal enzymes due to the test item, as it is commonly seen with chemicals (Greaves, 2012).
In view of the low magnitude of this finding and in the absence of degenerative associated process, the liver hypertrophy was not considered as adverse under the conditions of this study, but rather an adaptive change.
Altogether, the liver findings seen in cohort 1A were similar to those recorded in P generation, but they were noted with lower severity.
At the quantitative evaluation of the ovarian corpora lutea or primordium follicles in the P generation and cohort 1A, no statistically significant differences could be evidenced when compared to controls.
There were no alterations in the reproductive organs from females from both generations (ovaries, uterus, vagina), in agreement with the evaluation of the corresponding vaginal smears performed the day of necropsy.

Effect levels (P1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
Remarks on result:
other: Without test item-related findings in the brain
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive/ developmental toxicity
Effect level:
< 5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Remarks on result:
other: Was not associated with maternal neurotoxicity which was only observed high dose group

Target system / organ toxicity (P1)

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
25 mg/kg bw/day (actual dose received)
System:
nervous system
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
5 mg/kg bw/day (actual dose received)
System:
female reproductive system
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
From 5 mg/kg bw/day onwards and when compared with controls, F1 pups had finding at gross external examination (scab on nose, emaciated appearance, generalized pallor, absence of milk in stomach, hematoma on head) and qualitative assessment of body temperature (cold of the touch). These findings were associated with low birth and Day 4 p.p. survival index. Therefore, they were considered to be test item-related and adverse.
From 5 mg/kg bw/day onwards, some pups were cold to the touch, had dehydration and/or generalized pallor, mainly on Day 1 p.p. These findings were considered to be the consequence of lack of maternal care and, test item treatment related and adverse. The other findings listed in the above table were recorded in both controls, observed at low incidences with no dose-level relationship and/or are common findings in this species and strain maintained in the experimental conditions of this study.

Cohort 1A
Ptyalism was observed in all test item-treated groups (males and females) and with increased incidences. This finding is commonly observed after gavage and was considered to be test item treatment-related but not adverse.
Other findings observed with a low and/or similar incidence across groups (including controls) are those that are commonly observed in this species/strain of animals or in pregnant animals, and they were therefore not considered to be test item treatment-related.

Cohort 1B
Clinical signs recorded in 1B Cohort males and females are presented in Table 10 and Table 11, respectively of the section "Any other information on results incl. tables". At 25 mg/kg bw/day and in both sexes, there was a series of clinical signs suggestive of neurologic disorders (e.g. clonic/tonic convulsion, loss of balance and/or staggering gait) which were observed mainly from Study Day 94 after dosing for 1 to 3 days. Taking into account the nature and incidences of these finding, these observations were considered to be test-item-related and adverse.
Ptyalism was observed with an increased incidence in test item-treated groups when compared with controls. This finding is commonly observed after gavage and was therefore considered to be test item treatment-related but not adverse. Other findings observed with a low and/or similar incidence across groups (including controls) are those that are commonly observed in this species/strain of animals or in pregnant animals, and they were therefore not considered to be test item treatment-related.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
There was no mortality in non-selected pups.

The repartition of deaths in F1 lactating pups (%) and the macroscopic post-mortem observations in found dead pups are summarized Table 5 and 6, respectively in the section "Any other information on results incl. tables".
From 5 mg/kg bw/day and when compared with controls, there was a statistically significant higher percentage of pups found dead, missing and/or cannibalized on Days 1- 4 p.p. (12.5, 16.2 and 32.3% respectively at 5, 15 and 25 mg/kg bw/day vs. 4.9% in controls). At necropsy of found dead pups, absence of milk and autolysis were noted in all groups including control group. The incidences of both findings were increased in treated groups and dose-related. Absence of milk in the stomach may represent nursing difficulties or absence of maternal care. These findings were considered to be test item treatment-related and adverse from 5 mg/kg bw/day.

The viability and lactation indexes in F1 lactating pups (%) are summarized Table 7, respectively in the section "Any other information on results incl. tables". Before culling on Day 4 p.p. and when compared with controls or Historical Control Data, there was a lower Day 4 p.p. viability index (down to 74.4% vs. 94.8% in controls or 98.0% in HCD) at all doses. This finding was considered to be test item treatment-related and adverse. After culling on Day 4 p.p., there were no effects on lactation index.

Cohort 1A
There were no unscheduled deaths in Cohort 1A animals.

Cohort 1B
There were no unscheduled deaths in males. In females, there was one animal in the high dose group found dead on study day 1, without showing adverse clinical signs the day before death. Additionally, 3 animals were euthanized in the control group, 0 in the low dose group, 3 in the mid dose group and 5 in the high dose group. Two of the euthanized animals in the mid dose group and all of the euthanized animals in the high dose group were considered to be test item-related. In the mid dose group, 2 females were euthanized for dead litter on Day 1 p.p. and one female for no delivery (not pregnant). In the high dose group, 2 females were euthanized for dead litter on Day 1, one female because of lateral recumbency and loss of balance, one female because of hypoactivity, lateral recumbency and clonic convulsion, and one female because of difficulties to deliver on Day 24 p.c.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In lactating F1 pups, at 25 mg/kg bw/day and when compared with controls, mean body weight was low in males on Day 1 p.p. (-8%, p<0.01) with a tendency towards a return to control values thereafter. Therefore, while a test item-relationship could not be excluded, this finding was of minimal amplitude and considered to be non-adverse. At 15 and 5 mg/kg bw/day, there were no test item treatment-related effects on body weight and body weight gain.

Cohort 1A
There were no adverse effects on mean body weight or mean body weight changes. When compared with controls, the few statistically significant differences were recorded without any dose level relationship and with minimal amplitudes. Therefore, any test item treatment-related effect was considered to be unlikely.

Cohort 1B
During the pre- (males and females) and post-mating (males) periods, there were no adverse effects on mean body weight or mean body weight change in Cohort 1B animals. The few statistically significant differences (recorded without a dose level relationship and with minimal amplitude when compared with controls) were considered to be fortuitous.
During the pregnancy period, there were no adverse effects on mean body weight or mean body weight change in Cohort 1B females. At 15 and 25 mg/kg bw/day, statistically significant increases in body weight were observed. They were of minimal amplitudes when compared with controls and considered to be fortuitous during the pregnancy period (these increased mean body changes were mainly gained during the pre-mating period).
During the lactation period, there were no adverse effects on mean body weight or mean body weight change in Cohort 1B females. The few statistically significant differences were of minimal amplitude when compared with controls and with no dose level relationship. Therefore, they were considered to be fortuitous.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A
There were no effects on mean food consumption.

Cohort 1B
During the pre- (males and females) and post-mating (males) periods, there were no test item treatment-related effects on mean food consumption in Cohort 1B animals. The few statistically significant differences were of minimal amplitude, observed without any dose level relationship and therefore considered to be fortuitous.
During the pregnancy period, no adverse effects on mean food consumption in Cohort 1B females. The statistically significant difference was isolated and of minimal amplitude. Therefore, this difference was considered to be fortuitous.
During the lactation period, there were no significant effects on mean food consumption in Cohort 1B females during the lactation period.
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):
Cohort 1A
There were no test item-related effects. A statistically significant difference was recorded in low-dose male group (large unstained cells (LUC): 0.19 vs. 0.12 G/L in controls, p<0.01). There was no similar finding in females, no dose response and the value remained within the range of Historical Control Data. Therefore, a test item-relationship was considered to be unlikely.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A
There were no test item treatment-related effects on blood biochemistry in Cohort 1A animals. The statistically significant differences in Cohort 1A animals were recorded with no dose relationship, were not observed in both sexes and/or remained within the range of HCD. Therefore, a test item-relationship was considered to be unlikely.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A
From 15 mg/kg bw/day onwards in males and at 25 mg/kg bw/day in females, there were increases in mean urinary volume when compared with controls (+33% in males, down to p<0.01 and +27% in females, not statistically significant). Taking into account the low amplitude of the changes, this finding was considered to be non-adverse.
Sexual maturation:
no effects observed
Description (incidence and severity):
Cohort 1A
There were no effects on mean age at balanopreputial separation or on mean body weight on the day of occurrence in Cohort 1A males. There were no effects on mean age at vaginal opening or on mean body weight on the day of occurrence in Cohort 1A females. In the 5 mg/kg bw/day group, one female did not had a complete vaginal opening until euthanasia. This female had a fine membranous vaginal remnant (a common observation in this strain) which is known to have no impact mating success. There were no effects on mean time (number of days) to first estrous after vaginal opening in Cohort 1A females.

Cohort 1B
There were no effects on the mean age of balanopreputial separation or on mean body weight on the day of occurrence in Cohort 1B males. In the 5 and 25 mg/kg bw/day groups, two males had an incomplete preputial separation until euthanasia. This is a common finding in this species and strain for which a test item-relationship was considered to be unlikely.
There were no effects on the mean age at vaginal opening or on mean body weight on the day of occurrence in Cohort 1B females.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
In the F1 pups, there were no effects on Day 1 p.p mean anogenital distance (AGD) and normalized AGD, both in males and females.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
In the F1 pups, there were no nipples or areolae in male pups examined on Day 12 p.p.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were not test item-related organ weight differences in non-selected pups. The few organ weight changes were not considered to be test item-related because they were of insufficient magnitude and/or were not dose-related.

Cohort 1A
Increased absolute and relative-to-body liver weights were recorded in females treated at = 5 mg/kg bw/day and in relative-to-body weights in males treated at 25 mg/kg bw/day (up to +18%; p<0.01 or 0.05). This correlated with hepatocellular hypertrophy at microscopic examination.
Increased absolute and relative-to-body mesenteric lymph node weights were recorded in females treated at = 15 mg/kg bw/day (up to +30%; p<0.01 or 0.05). There were no microscopic correlates. The relationship to test item administration was thus considered to be unlikely.
The other organ weight changes were not considered to be test item related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings.

Cohort 1B
Increased absolute and/or relative-to-body liver weights were recorded in males and females treated at = 15 mg/kg bw/day.
The other organ weight changes were not considered to be test item-related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings. This included the absolute and relative-to-body kidney weights that were minimally increased in females treated at 15 mg/kg bw/day when compared to controls. In the absence of dose-relationship and of similar differences in the other cohorts, this change was considered to be unrelated to the test item administration.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no test item-related gross changes in non-selected pups. The few other isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age.

There were no test item treatment-related macroscopic post-mortem findings in any groups in F1 pups euthanized after weaning.

Cohort 1A
There was no unscheduled mortality. There were no test item-related findings at macroscopic post-mortem examination. The few isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age. This included the black discoloration seen in the stomach from three high-dose males (correlated generally with background erosion), that was seen also in low- and mid-dose females.

Cohort 1B
The few isolated gross observations at macroscopic post-mortem examination were considered to be consistent with spontaneous findings encountered in the rats of these strain and age. This included the thickened uterus that correlated with remnants of gestational glands in several females treated at 15 or 25 mg/kg bw/day, and the black mass located in the uterus of 2 out of 15 females treated at 25 mg/kg bw/day. This was not associated with reproduction trouble and correlated with placental remnants bearing necrosis and hemorrhage at microscopic examination.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A
Test item-related changes were noted in the liver and consisted in non-adverse dose-related minimal hepatocellular hypertrophy in males (1 out of 20 animals, grade 1) and females (14 out of 2 animals, grade 1) treated at 25 mg/kg bw/day. The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.
There were test item-related changes neither in the male reproductive organs, including the detailed examination of the testes using a thorough understanding of tubule development through the different stages of the spermatogenic cycle, or in the female reproductive organs.
There was a good correspondence between the vaginal smears and the histopathological examination of estrus cycle in all examined females.

At enumeration of corpora lutea in the ovary of high-dose and control groups, there were no differences, with a mean number of corpora lutea of 18.45 (±6.60) and 19.00 (±7.36) per animal respectively, reaching no statistical significance. The mean number of primordial follicles was lower in females treated at = 5 mg/kg bw/day when compared to the control group (8.68±6.77 in the low dose group versus 9.31±6.09 in the control group). However, these differences did not reach statistical significance and did not correlate with any microscopic changes in the reproductive system. Thus, these differences were considered to be unrelated to the test item administration.

Cohort 1B
Slight degeneration/necrosis in placenta with hemorrhage, accumulation and/or mineralization was seen in 2 out of 20 females treated at 25 mg/kg bw/day. There was also minimal focal hemorrhage in the uterus from one female treated at 15 mg/kg bw/day. These females had no reproduction troubles. The relationship to test item was considered to be unlikely, in the absence of microscopic examination of the control group from this cohort, and of the absence of similar changes in the cohort 1A.
In the other examined organs, there were no test item-related findings, including in the brain with dedicated examination.
The few microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity
Generation:
F1 (cohort 1B)
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive/developmental toxicity
Generation:
F1 (cohort 1B)
Effect level:
< 5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other:
Remarks on result:
other: Was not associated with maternal neurotoxicity which was only observed in the high dose group

Target system / organ toxicity (F1)

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
25 mg/kg bw/day (actual dose received)
System:
nervous system
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
5 mg/kg bw/day (actual dose received)
System:
female reproductive system
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: F2 generation

General toxicity (F2)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
From 15 mg/kg bw/day, there was a dose-related increase in the number of pups with findings on Day 1 p.p.
The qualitative appreciation of body temperature (or cold to the touch) was mainly concerned from 15 mg/kg bw/day and/or with emaciated appearance and hypoactivity at 25 mg/kg bw/day. These findings were considered to be test-item treatment related and adverse. Other findings observed in lactating pups are commonly observed in this species/strain of animals at this age.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
There were dose-related increase incidences of found dead and/or cannibalized pups in all test item-treated groups (3.4%, 24.2% and 37.4% at 5, 15 and 25 mg/kg bw/day vs. 1.0% in controls, respectively; with p<0.001 from 15 mg/kg bw/day). These findings were considered to be test item-related and adverse.
From 15 mg/kg bw/day and when compared with controls, there were lower viability indexes (80.6% and 56.6% at 15 and 25 mg/kg bw/day respectively vs. 99.0% in controls or 93.1 % in Historical Control Data). This finding was considered to be test-item related and adverse.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 25 mg/kg bw/day and when compared with controls, male lactating F2 pups had a low body weight at Day 1 p.p. (-9.9%, p<0.01, when compared with controls) with the same tendency in female pups (-6.5%, not statistically significant). There were no effects on body weight changes between Day 1 p.p. and Day 4 p.p. Taking into account the amplitude of the change of body weight and the slight recovery observed at Day 4 p.p., this finding was considered to be test item treatment-related but non adverse. At 15 and 5 mg/kg bw/day, when compared with controls, there were no adverse effects on the mean body weight and body weight change during lactation.
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:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
At 25 and 15 mg/kg bw/day, absence of milk in the stomach may represent nursing difficulties or absence of maternal care. From 5 mg/kg bw/day, there was a dose-related increased number of pups found dead (autolysis). These findings were considered to be test item treatment-related and adverse. All findings recorded in euthanized as scheduled F2 pups are commonly observed in this species/strain of animals at this age.
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity
Generation:
F2
Effect level:
< 5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
5 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

CHEMICAL ANALYSIS OF THE DOSE FORMULATIONS


The test item concentrations in the administered dose formulations analyzed in premating, mating, gestation and lactation periods of the P-generation and in postweaning, premating, gestation and lactation periods of the F1 generations remained within an acceptable range of variations (-5.3% to +5.1%) when compared to the nominal values (± 15% of the nominal concentrations). No test item was observed in the control dose formulation.

Applicant's summary and conclusion

Conclusions:
An Extended One-Generation Reproductive Toxicity Study (EOGRTS) was performed with 1,3-diphenylguanidine, by the oral route (gavage) in rats. The study was performed in compliance with GLP and according to OECD Guideline No. 443 and ECHA decision CCH-D-2114460631-54-01/F.
The test item was administered daily at the dose level of 0, 5, 15 or 25 mg/kg bw/day to sexually-mature male and female rats (parental (P) generation), continuously from 10 weeks before mating and through mating, gestation and weaning of the pups (F1 generation). At weaning, pups were also treated daily by oral gavage at the dose level of 0, 5, 15 or 25 mg/kg bw/day. Pups were assigned to cohorts for reproductive/developmental toxicity testing (Cohorts 1A and 1B, including the production of a F2 generation).

Systemic toxicity evaluation:
The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 15 mg/kg bw/day based on the severe clinical signs suggestive of neurologic disorders in males and females, leading to premature euthanasia in females at 25 mg/kg bw/day.

Reproductive/developmental toxicity evaluation:
The No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was considered to be lower than 5 mg/kg bw/day based on the increased gestation periods at all dose-levels, reproductive troubles in females (found dead litters from 5 mg/kg bw/day, difficulties to deliver from 15 mg/kg bw/day) resulting in high incidence of pup mortality at birth and on the first days of lactation from 5 mg/kg bw/day, not associated with maternal neurotoxicity which was only observed at the top dose-level of 25 mg/kg bw/day.
Executive summary:

The objective of this study was to provide general information concerning reproductive and developmental effects of the test item, 1,3-Diphenylguanidine, following daily oral administration (gavage) that may occur as a result of pre- and post-natal exposure as well as an evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring.


The dose formulations were administered to sexually-mature male and female rats (Parental (P) generation) by oral gavage, at test item dose levels of 0, 5, 15 or 25 mg/kg/day, daily, starting 2 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, pups were also treated daily by oral gavage, at test item dose levels of 0, 5, 15 or 25 mg/kg/day.


Pups were assigned to Cohorts for reproductive/developmental toxicity testing (Cohorts 1A and 1B, including the production of an F2 generation),


Methods


Treatment


P generation and F1 lactating offspring:


Three groups of 24 male and 24 female Sprague-Dawley rats (P generation) received the test item, 1,3-Diphenylguanidine (batch No. 18051468027), daily for 2 weeks prior to pairing, during pairing, through gestation and lactation until weaning of the F1 pups [Day 21 post-partum (p.p.)]. The test item was administered orally (gavage, 5 mL/kg). A control group of 24 males and 24 females received the vehicle alone (0.5% methylcellulose in drinking water treated by reverse osmosis), under the same experimental conditions, and acted as a reference control group.


F1 generations:


The test item was administered orally, by gavage (5 mL/kg) to groups of 20 rats/sex (Cohorts 1A and 1B) receiving 0 (0.5% methylcellulose in drinking water treated by reverse osmosis), 5, 15 or 25 mg/kg/day. The treatment schedules were the following:



  • Cohort 1A: daily from weaning (Day 22 p.) until euthanasia (on Days 92 to 96 p.p.).

  • Cohort 1B: daily from weaning (Day 22p.) until euthanasia. Males were treated from weaning (Day 22 p.p.) for at least 10 weeks before mating, during the mating period (up to 2 weeks) and after euthanasia of F2 pups (on Day 4 p.p.). Females were treated from weaning (Day 22 p.p.) for at least 10 weeks before mating, during the mating period (up to 2 weeks), during gestation, during lactation (until Day 4 p.p. inclusive) or until euthanasia for females with no evidence of mating or no delivery (24 days after the last day of the mating period).


Examination of Parental, F1 and/or F2 generations


Clinical signs and mortality were checked daily. Food consumption and body weight were recorded at designated intervals.


P generation and Cohort 1B males and females were paired until mated or until 14 days had elapsed.


Females from the P generation and Cohort 1B were allowed to deliver normally and rear their progeny. Pregnancy and litter parameters were recorded.


During lactation, the F1 and F2 pups were observed daily for survival and clinical signs. Body weight was measured at designated intervals and the sex-ratio was recorded. In F1, the size of each litter was adjusted on Day 4 p.p. to obtain ten pups per litter. Pup physical and/or reflex development was assessed at designated time-points.


Examination of Cohorts


Cohort 1A: animals were selected for assessment of general toxicity and effects on their reproductive system. Estrous cycle stages were determined daily for all females after the onset of vaginal patency, until the first cornified smear was recorded (estrus), and for 19 days before the end of the treatment period.


Cohort 1B: animals were selected for follow-up assessment of reproductive performance (by mating F1 animals) and to potentially obtain additional histopathology data.


Terminal examinations


A macroscopic post-mortem examination was performed on all P and F1 animals (including F1 pups culled on Day 4 p.p. and F1 pups not selected on Day 22 p.p.) and F2 pups. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was given to the reproductive organs. The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated and organs were weighed wet as soon as possible after dissection. A microscopic examination was performed on all macroscopic lesions and a complete list of tissues from animals in all groups or the control and high-dose groups.


P generation and Cohort 1A: the first 10 surviving animals/sex/group were fasted (food only) for an overnight period of at least 14 hours prior to blood sampling (for hematology, coagulation and blood biochemistry) and urine collection (for urinalysis) at termination. A series of sperm cell evaluations was performed on surviving males from all groups or the control and high-dose groups: motility, morphology, sperm cell head count in testicular/epididymal tissues.


Cohort 1A: splenic lymphocyte subpopulation analysis was performed on 10 surviving animals/sex/group from Cohort 1A.


Thyroid hormone levels (P generation, F1 culled pups, Cohort 1A animals and F2 pups): prior to blood sampling the animals were not fasted (except for animals from which samples were collected for hematology, blood biochemistry and urinalysis). Blood samples were taken on Day 4 p.p. (F1 and F2 pups) for measurements of thyroid hormone (T4) levels, and on Day 22 p.p. (F1 pups not selected for Cohorts) and at termination from the first ten surviving males/group and the first ten lactating females/group (P and Cohort 1A animals), for measurements of thyroid hormone (T4) and Thyroid Stimulating Hormone (TSH) levels.


Results


The test item concentrations in the administered dose formulations analyzed during each period (P-premating, P-mating, P-gestation, P-lactation, F1-postweaning, F1-premating, F1-gestation and F1-lactation) remained within an acceptable range of variations (-5.3% to +5.1%) when compared to the nominal values (± 15% of the nominal concentrations). No test item was detected in the control dose formulation.


P generation


Mortality: there were no unscheduled deaths in males. In pregnant females and from 5 mg/kg/day, there were premature deaths considered to be test item related. Namely two females were sacrificed for humane ground at 25 mg/kg/day, two were sacrificed due to the difficulties to deliver (one at 15 mg/kg/day and one at 25 mg/kg/day). In addition, five females (one at 5 mg/kg/day, one at 15 mg/kg/day and three at 25 mg/kg/day) were sacrificed due to the death of their litters.


Clinical signs: in both sexes, there were dose-related increases in ptyalism that were considered to be test item-related, but not adverse as this is a common finding observed after oral gavage. At 25 mg/kg/day and in females at the end of the pregnancy period (around parturition), there was a series of clinical signs suggestive of neurologic disorders (clonic convulsion, locomotory difficulties, loss of balance, staggering gait and/or tonic seizures) which were transient and observed after dosing. These findings were considered to be test-item treatment related and adverse.


Mean body weight, mean body weight change and mean food consumption: there were no adverse effects during the premating, mating, gestation or lactation periods.


Estrous cycle, mating and fertility: there were no adverse effects on estrous cycle, mating (including the mean number of days taken to mate) or fertility.


Gestation: there was a trend towards increased gestation periods in test-item treated females. Two females at 5 mg/kg/day had 24-day gestation periods associated with high pup mortality rates (90-100%). At 25 mg/kg/day, there was an increased number of females with 23-day gestation periods associated with high post-implantation losses (27.1 vs. 15.6% in controls, p<0.05). This finding also observed in Cohort 1B was considered to be test item treatment-related and adverse.


Delivery data: there was a dose-related tendency towards lower number of viable pups from 5 mg/kg/day, despite an absence of statistically significant difference vs. controls or when compared with Historical Control Data. At 25 mg/kg/day, on Day 1 p.p, the number of viable pups was down to 9.3 vs. 12.0 in controls and live birth index down to 72.8 vs. 95.1% in controls. These findings were considered to be test item treatment-related and adverse.


P generation offspring (pre-weaning F1 pups): from 5 mg/kg/day and when compared with controls, there was a statistically significant higher percentage of pups found dead, missing and/or cannibalized on Days 1-4 p.p. (12.5, 16.2 and 32.3%, respectively at 5, 15 and 25 mg/kg/day, vs. 4.9% in controls, with p<0.01 or <0.001). At necropsy of found dead pups, absence of milk and autolysis were noted with high incidence in the treated groups when compared to the control group. Absence of milk in the stomach may represent nursing difficulties or absence of maternal care. These findings were considered to be test item treatment-related and adverse.


Laboratory investigations: there were no test item treatment-related effects on hematology, coagulation, blood biochemistry or urinalysis in P generation animals. In P generation females and at 25 mg/kg/day, when compared with controls or Historical Control Data, there were high mean T4 concentration (+38%, p<0.05) associated with low mean TSH concentrations (-14%). Taking into account the amplitude of the change and the association of the effects, a test item-relationship cannot be excluded but considered to be non-adverse in the absence of finding at pathology examination. There were no effects at 15 and 5 mg/kg/day.


Sperm analysis: there were no effects on sperm analysis parameters (motility, morphology, sperm/testicular numerations or daily sperm production rate).


Cohort 1A


Mortality: there were no unscheduled deaths.


Clinical signs: ptyalism was observed in all groups (including controls) but with an increased incidence in test item-treated groups when compared with controls. This finding is commonly observed after gavage and was considered to be test item treatment-related but not adverse.


Body weight, body weight change and food consumption: there were no adverse effects.


Sexual development: there were no effects.


Estrous cycles: there were no effects on mean time to first estrous after vaginal opening and on mean estrous cycle parameters.


Laboratory investigations: there were no adverse test item treatment-related effects on hematology, coagulation, blood biochemistry or urinalysis in P generation animals. There were no effects on mean thyroid hormones levels (T4 and TSH).


Sperm analysis: there were no effects on sperm analysis parameters (motility, morphology, sperm/testicular numerations or daily sperm production rate).


Lymphocyte subtyping (Cohort 1A): there were no test item treatment-related findings.


Cohort 1B


Mortality: there were no unscheduled deaths in males. In pregnant females and from 15 mg/kg/day, there were premature deaths considered to be test item related. Namely two females were sacrificed for humane ground at 25 mg/kg/day, one was sacrificed due to the difficulties to deliver at 25 mg/kg/day and four were sacrificed due to the death of their litters (two at 15 mg/kg/day and two at 25 mg/kg/day)..


Clinical signs: in both sexes, there were dose-related increases in ptyalism that were considered to be test item-related, but not adverse as this is a common finding observed after oral gavage. At 25 mg/kg/day and in both sexes, there was a series of clinical signs suggestive of neurologic disorders (e.g. clonic/tonic convulsion, loss of balance and/or staggering gait) which were observed after dosing and considered to be test item-related and adverse.


Body weight, body weight change and food consumption: there were no adverse effects on mean body weight, mean body weight change or mean food consumption.


Sexual development: there were no effects.


Estrous cycles, mating and fertility: there were no effects.


Gestation: at 25 mg/kg/day, mean duration of gestation was increased (22.3 vs. 21.9 days in controls, p<0.05). This finding was considered to be test-item related (already observed in P generation) and adverse. There were dose-related increased post-implantation losses from 5 mg/kg/day.


Delivery: as a consequence of increased post-implantation losses, there were lower live birth indexes at 15 and 25 mg/kg/day (84.7% and 67.5% vs. 100.0% in controls, respectively) and lower number of live pups at 25 mg/kg/day (7.8 vs. 11.4 in controls, p<0.01). These findings were considered to be test-item related and adverse.


F1 generation offspring (pre-weaning F2 pups): from 15 mg/kg/day and when compared with controls, there were low viability indexes on Day 4 p.p. (down to 56.6% at 25 mg/kg/day vs. 99.0% in controls or 93.1 % in Historical Control Data). At external examination, this finding was associated with pups cold to the touch from 5 mg/kg/day and/or with emaciated appearance at 25 mg/kg/day. These findings were considered to be test-item related and adverse. They were considered to represent lack of maternal care as confirmed with the increased number of found dead pups with autolysis and/or absence of milk in the stomach. There were no deaths after Day 4 p.p. in pre-weaning F2 pups.


Pathology


P generation: There was a dose-related increased incidence of mortality in test item-treated females at ≥ 15 mg/kg/day due to reproductive trouble, and at 25 mg/kg/day with not evident cause of death. Increased liver weights were recorded in males treated at ≥ 15 mg/kg/day and correlated with the microscopic hepatocellular hypertrophy.  There were no gross test item-related findings. Dose-related minimal non-adverse hepatocellular hypertrophy was noted in the liver in males and females treated at 25 mg/kg/day. At quantitative evaluation of primordium follicles or corpora lutea, there were no differences between the high-dose and the control groups.


Cohort 1A: There was no mortality. Increased liver weights were recorded in females treated at ≥ 5 mg/kg/day and in males treated at 25 mg/kg/day. This correlated with hepatocellular hypertrophy at microscopic examination. There were no gross test item-related findings. Dose-related minimal hepatocellular hypertrophy was noted in the liver from in males and females treated at 25 mg/kg/day. At quantitative evaluation of primordium follicles or corpora lutea, there were no differences between the test item-treated groups and the controls.


Cohort 1B: Test item-related mortality was noted at 15 and 25 mg/kg/day. Reproduction trouble (dead litter or difficulties to deliver) were noted for two females treated at 15 mg/kg/day and for three females treated at 25 mg/kg/day. No evident cause could be established for two females treated at 25 mg/kg/day. Increased liver weights were recorded in males and females treated at ≥ 15 mg/kg/day. No microscopic findings were considered to be test item-related (examination of brain and macroscopic findings).


Non-selected pups: There was no mortality. There were not test item-related organ weight differences. There were no test item-related gross changes.


Conclusion


The test item, 1.3-Diphenylguanidine, was administered daily by oral gavage, at dose levels of 0, 5, 15 or 25 mg/kg/day, to sexually-mature male and female rats (parental (P) generation) starting 2 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, the F1 generation was also exposed to graduated doses of the test item and was assigned to Cohorts of animals for reproductive/developmental toxicity.


Systemic toxicity evaluation:


The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 15 mg/kg/day based on the severe clinical signs suggestive of neurologic disorders in males and females, leading to premature euthanasia in females for humane grounds at 25 mg/kg/day.


Reproductive/developmental toxicity evaluation:


The No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was considered to be lower than 5 mg/kg/day based on the increased gestation periods at all dose-levels, reproductive troubles in females (found dead litters from 5 mg/kg/day, difficulties to deliver from 15 mg/kg/day) resulting in high incidence of pup mortality at birth and on the first days of lactation from 5 mg/kg/day, not associated with maternal neurotoxicity which was only observed at the top dose-level of 25 mg/kg/day.