Trimethoxyvinylsilane

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity – with F2 generation and developmental immunotoxicity (Cohorts 1A, 1B with extension, and 3)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 November 2019 to 21 October 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

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:

- Premating exposure duration for parental (P0) animals : 2 weeks as requested in ECHA Final Decision No CCH-D-2114405896-41-01/F.
- Basis for dose level selection : Based on the findings of a 28-day dose range finding study (BSL Bioservice, 2020) and a combined repeated dose and reproduction/developmental toxicity screening test (Hashima Laboratory, 2005) with trimethoxy(vinyl)silane. The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering.
- Inclusion/exclusion of extension of Cohort 1B: Included, because it was requested in ECHA Final Decision No CCH-D-2114405896-41-01/F.
- Termination time for F2 : F2 animals were terminated at weaning (PND 22) according to OECD Test Guideline 443.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B : Not included, because they were not requested in ECHA Final Decision No CCH-D-2114405896-41-01/F and based on no evidence of neurotoxicity in the available data for trimethoxy(vinyl)silane.
- Inclusion/exclusion of developmental immunotoxicity Cohort 3 : Included, because Cohort 3 was requested in ECHA Final Decision No CCH-D-2114405896-41-01/F.
- Route of administration : The oral gavage route was selected based on test item characteristics and stability.

Test material

Test material information

Constituent 1

Reference substance name:

Trimethoxyvinylsilane

EC Number:

220-449-8

EC Name:

Trimethoxyvinylsilane

Cas Number:

2768-02-7

Molecular formula:

C5H12O3Si

IUPAC Name:

ethenyltrimethoxysilane

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han) (Full Barrier)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 12-13 weeks old; (F1) x wks
- Weight at study initiation: (P) Males: 268 - 317 g; Females:163 - 206 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: no
- Housing:
Pre-mating period of P and F1: male and female animals were housed in groups of 5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages.
Mating period: During mating period males and females (Parental and F1) were housed together in ratio 1:1 (male to female)
Post-mating period: during post-mating period males (Parental and F1) were housed in groups of 5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages.. After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages.
- Diet (e.g. ad libitum): Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3°C
- Humidity (%): 55+/-10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

IN-LIFE DATES: From: 6th November 2019 To: 3rd April 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulations were vortexed and/or stirred until visual homogeneity was achieved.
After homogenization the formulation was overlaid with argon to prevent hydrolysis caused by contact of the test item formulation with water.
Based on the results of stability testing, the test item formulations were prepared at least every 12 days. The prepared formulations were stored at room temperature and overlaid with argon.


VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle for test item formulations had been selected in consultation with the sponsor based on the test item’s characteristics.
- Concentration in vehicle: 0, 10, 25, 75 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no. (if required): MKCH1635

Details on mating procedure:

- M/F ratio per cage:
Parental animals: 1:1 pairing
Cohort 1B animals: 1:1 pairing
- Length of cohabitation: until pregnancy occurred or up to 2 weeks
- Proof of pregnancy: vaginal sperm or a vaginal copulation plug referred to as day 0 of pregnancy
- In case mating had not occurred after 2 weeks, the animals were separated without further opportunity for mating.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Stability analysis: conducted before the study start on the samples from HD and LD groups and the investigation was made for 0 h, 6 h (RT), 12 days (RT), 12 days (2 to 8 °C) and 11 days (-15 to -35 °C). The test item formulations were stable for up to 12 days at room temperature.
- Homogeneity: conducted before the study start on the samples collected from various levels (top, middle and bottom) of HD and LD groups. Based on the results from the setup the test item formulation was a suspension. The test item was shown to be homogenous after 30 min without stirring, samples were not collected during the study for the investigation of homogeneity.
- Concentration analysis: duplicate samples were taken for substance concentration analysis. Concentration analysis of formulation samples was performed at three concentrations, 10.00 mg/mL, 25.00 mg/mL and 75.00 mg/mL in study weeks 1, 5, 9, 13 and in the last week of the study.

Duration of treatment / exposure:

- Parental males: dosed until the minimum total dosing of 10 weeks, i.e. during 14 days of pre-mating, maximum 14 days of mating and post-mating until terminal sacrifice.
- Parental females: dosed during 14 days of pre-mating, maximum 14 days of mating, during gestation and lactation until weaning (PND 21).
- F1 offspring: received treatment with the test item from weaning (PND 21) to terminal sacrifice of the respective cohort.
- Cohort 1A animals (F1 generation): treated from weaning to week 13 of age (10 weeks treatment), sacrifice at the age of 13 weeks
- Cohort 1B animals (F1 generation): treatment until weaning of the F2, or until termination of the study (week 20 - 25)
- Cohort 3 animals (F1 generation): treatment from weaning to week 8-10 of age (5-7 weeks treatment), sacrifice at the age of 8 -10 weeks.

Frequency of treatment:

daily

Details on study schedule:

- F1 parental animals not mated until PND 90 after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 90 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control group (corn oil); Group 1

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Remarks:

Low dose group (LD); Group 2

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Mid dose group (MD); Group 3

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

High dose group (HD); Group 4

No. of animals per sex per dose:

- Parental animals: 25 male and 25 female animals per group
- Cohort 1A: 20 M + 20 F / Group
- Cohort 1B: 20 M + 20 F / Group
- Cohort 3: 10 M + 10 F / Group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In consultation with the sponsor and based on the results of a 28-day dose range-finding study (BSL Bioservice, 2019). The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and NOAEL. In the dose range finding study male and female Wistar rats were treated with the test item at doses of 0 (vehicle), 50, 150, 600 and 1000 mg/kg bw/day. No mortality or marked clinical signs and no effects of the test item on functional observations, body weight development, food consumption, clinical biochemistry or urinary parameters were observed up to the highest tested. Test item-related increases in haematological parameters were observed in reticulocytes of males at =600 mg/kg bw/day and females at =150 mg/kg bw/day and in monocytes of males and females at =150 mg/kg bw/day. Test item-related lesions were observed in the kidneys (enlargement, renal pelvis dilatation), ureters (dilatation) and urinary bladder (thickened wall). Adverse histopathological treatment-related changes were observed in organs of the urinary system (i.e. urothelial hyperplasia, inflammatory reactions, liminal precipitate(s), granular casts, tubular dilatation, and tubular basophilia) in animals at =150 mg/kg bw/day, and in the small intestine with regional lymphoid tissues (lipid accumulation) in animals at =600 mg/kg bw/day.
In a combined repeated dose and reproduction/developmental toxicity screening test (Hashima Laboratory, 2005, OECD 422) male and female Sprague-Dawley rats were treated with the test item at doses of 0 (vehicle), 62.5, 250, and 1000 mg/kg bw/day. Two males and 1 female from the 1000 mg/kg group died on days 16 and 17 (males) and day 8 (female). Transient salivation, soiled hair, a decrease in locomotor activity, reddish urine, hypothermia, perioral smudges, perianal soiling, diarrhoea, bradypnea, and piloerection were noted in the dying animals. Transient salivation, soiled hair and reddish urine were noted in the surviving males and females at =250 mg/kg bw/day. Lower body weights were noted in males on days 4 to 42 during the administration period and days 1 and 4 during the recovery period. Females at 1000 mg/kg bw/day also showed a decrease in body weight.
In females, the number of oestrous cases before pairing at 1000 mg/kg bw/day was less than in the control group. In males, moderate atrophy of seminiferous tubule, slight degeneration of seminiferous tubule, slight vacuolization of Sertoli cell and slight retention of spermatid were observed at 1000 mg/kg bw/day. A slight decrease in sperm and slight or moderate cell debris were observed in lumen of the epididymis at 1000 mg/kg bw/day.
No changes were observed in reproductive parameters.
Body weight of males at 1000 mg/kg bw/day was increased after the administration period. No change of body weight was detected in males after the recovery period and no changes in body weight were detected in females after the administration period.
Decrease in absolute weight of thymus, increase in absolute weight of kidney, increase in relative weight of spleen, kidney and adrenals were observed in males at 1000 mg/kg bw/day after the administration period. Relative weight of thymus was decreased in females at 62.5 and 250 mg/kg bw/day. Decreased absolute weight of thymus, decreased relative weight of thymus and increased relative weight of liver were observed in females after the administration period.
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: yes, fasted overnight
- Cohort 1A animals (F1 generation): used to assess effect on reproductive system and toxicity; treated from weaning to week 13 of age (10 weeks treatment), sacrifice at the age of 13 weeks
- Cohort 1B animals (F1 generation): used to assess reproductive performance, additional histopathology data or endocrine toxicity; treatment until weaning of the F2, or until termination of the study (week 20 - 25)
- Cohort 3 animals (F1 generation): used to assess developmental immunotoxicity; treatment from weaning to week 8-10 of age (5-7 weeks treatment), sacrifice at the age of 8 -10 weeks.

Positive control:

Cohort 3: T-Cell Dependent Antibody Response Assay positive control group was administered oral gavage dose of 10 mg/kg bw/day cyclophosphamide (administration volume of 10 mL/kg bw) 7 days before immunization (i.e. PND 49 ± 3 days) until the day before the last blood sampling. Cyclophosphamide was diluted with 0.9 % sodium chloride to a concentration of 1 mg/mL.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: For Parental and selected F1 cohorts, general clinical observations of the animals were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. Twice daily all animals (Parental and F1 generation) were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily. Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality were recorded.
- Cage side observations included: health condition (once daily); morbidity and mortality (twice daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed clinical observations were made in all P animals and all F1 cohorts, when animals were weighed outside the home cage. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling, as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
Time schedule for examinations:
- Parental animals (P0): weighed once before the assignment to the experimental groups, on the first day of dosing and weekly during the study period as well as at the terminal sacrifice. In addition, during pregnancy, females were weighed on gestation days (GD) 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 and within 24 hours of parturition (day 0 post-partum) as well as on day 4, 7, 14 and 21 post-partum along with pups.
- F1 (all cohorts) animals: F1 animals were weighed every two days during the first 2 weeks following weaning, weekly thereafter and at termination. In addition, F1 females from Cohort 1B were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as on day 4, 7, 14 and 21 post-partum along with F2 pups. Body weight of all cohorts was also taken on the day of attainment of vaginal patency or balano-preputial separation. Body weight was also measured on PND 4, at weaning (surplus pups after selection of cohorts) and weekly until terminal sacrifice.

FOOD CONSUMPTION: Yes
Time schedule for examinations: Food consumption was measured at intervals corresponding to the body weight measurements after the beginning of the dose administration except during the mating period for Parental and Cohort 1B F1 animals. Food consumption was also not measured during the post-mating period in Parental and Cohort 1B F1 males until all females were mated and all males were back to their original housing cage of 5 males per cage.

HAEMATOLOGY
Time schedule for examinations: at the end of the treatment prior to sacrifice
How many animals: 10 randomly selected Parental males and females
Fasted overnight: yes
Parameters examined: see tables 3 & 4

CLINICAL BIOCHEMISTRY
Time schedule for examinations: at the end of the treatment prior to sacrifice
How many animals: 10 randomly selected Parental males and females
Fasted overnight: yes
Parameters examined: see table 5

THYROID HORMONE ANALYSIS
Time schedule for examinations: at the end of the treatment prior to sacrifice
How many animals: 10 randomly selected Parental males and females
Parameters examined: T4 & TSH

URINALYSIS
Time schedule for examinations: at the end of the treatment prior to sacrifice
How many animals: 10 randomly selected Parental males and females
Parameters examined: see table 6. Additionally, urine colour/appearance and volume were recorded. Additionally, microscopic analysis of urine for sediment and presence of blood and/or blood cells or cell debris was conducted.

Oestrous cyclicity (parental animals):

- Parental females: vaginal smears were examined 2 weeks before beginning of treatment period, during 2 weeks premating period and until the confirmation of mating and/or the end of the 2 weeks mating period to record the oestrous cyclicity and also to confirm the evidence of mating. The oestrous cycle stage was recorded in P at termination.
- F1 females in Cohort 1A: vaginal smears were examined daily for all animals after the onset of vaginal patency until the first cornified smear was recorded to determine the time interval between these two events. Vaginal smears in Cohort 1A were examined for 2 weeks starting from PND 75. The oestrous cycle stage was recorded in Cohort 1A at termination.
- F1 females in Cohort 1B: vaginal smears were examined during mating period to confirm the evidence of mating. The oestrous cycle stage was recorded in Cohort 1B at termination.
- Cohort 3: The oestrous cycle stage was recorded in Cohort 3 at termination.

Sperm parameters (parental animals):

Parameters examined in P and F1 (Cohort 1A and 1B) male generations: testis weight, epididymis weight
Parameters examined in P and F1 (Cohort 1A) male generations: sperm motility, sperm morphology, testicular sperm head count, cauda epididymis sperm count

Litter observations:

STANDARDISATION OF LITTERS
- On PND 21, F1 pups were randomly (with the exception of obvious runts) assigned between three Cohorts 1A (20 animals / sex / group), 1B (20 animals / sex / group) and 3 (10 males and 10 females per group; one per litter)
- The surplus pups at PND 4 were subjected to gross necropsy.
- The pups not selected for cohorts, including runts, were terminated after weaning, on PND 22 or when they were not needed for further in-life investigations.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
- Cage-side observations: Twice daily all animals (F1 and F2 generation) were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
- General litter observations: Each F1 and F2 litter was examined after parturition (PND 0) to establish the number and sex of pups, stillbirths, live births and the presence of gross external anomalies, including cleft palate, subcutaneous haemorrhages, abnormal skin colour or texture, presence of umbilical cord, lack of milk in stomach and presence of dried secretions. In addition, the first clinical examination of the neonates included a qualitative assessment of body temperature, state of activity and reaction to handling. Pups found dead on PND 0 or later time were examined for the possible cause of death. Live pups were counted and sexed.
- Litter weight: F1 and F2 litters were weighed within 24 hours of parturition (PND 0) or PND 1, on PND 4, 7, 14 and PND 21. In addition to the observations on Parental animals, any abnormal behaviour of the offspring was recorded.
- Clinical signs: The clinical signs of pups were recorded on the corresponding days when offspring were weighed.
- Anogenital distance (AGD) and pup body weight: AGD of each pup was measured once between PND 0 and PND 4. Pup body weight was measured on the day of the AGD measurement and was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD / Cube root of pup weight).
- Nipples/areolae retention: Male pups were checked for the presence of nipples/areolae on PND 12.
- Sexual Maturity: All selected F1 male and female pups from all cohorts (1A, 1B, and 3 with exception of surplus pups not selected for cohorts) were checked daily for balano-preputial separation or vaginal patency, respectively, starting from PND 30 in males and PND 25 in females. Any abnormalities of genital organs were recorded.

HAEMATOLOGY
Time schedule for examinations: at the end of the treatment prior to sacrifice
How many animals: 10 randomly selected F1 Cohort 1A males and females
Fasted overnight: yes
Parameters examined: see tables 3 & 4

CLINICAL BIOCHEMISTRY
Time schedule for examinations: at the end of the treatment prior to sacrifice
How many animals: 10 randomly selected F1 Cohort 1A males and females
Fasted overnight: yes
Parameters examined: see table 5

THYROID HORMONE ANALYSIS
Time schedule for examinations: at the end of the treatment prior to sacrifice
How many animals: 10 randomly selected F1 Cohort 1A males and females. Also performed on 10 pups/sex/group pups from Parental females on PND 4 and after weaning (pups not allocated to cohorts) on PND 22 or when they were not needed for further in-life investigations. Neonatal (PND 4) blood was pooled by litters for thyroid hormone analysis.
Parameters examined: T4 & TSH

URINALYSIS
Time schedule for examinations: at the end of the treatment prior to sacrifice
How many animals: 10 randomly selected F1 Cohort 1A males and females
Parameters examined: see table 6. Additionally, urine colour/appearance and volume were recorded. Additionally, microscopic analysis of urine for sediment and presence of blood and/or blood cells or cell debris was conducted.

GROSS EXAMINATION OF DEAD PUPS: Dead or moribund pups were recorded and examined for possible defects and/or cause of death and preserved.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Yes
- Details: On PND 56 ± 3 days, Cohort 3 animals were used in a T-cell dependent antibody response assay.
The positive control group (PC) was administered cyclophosphamide 7 days before immunization (i.e. PND 49 ± 3 days) until the day before the last blood sampling. Cyclophosphamide was diluted with 0.9 % sodium chloride to a concentration of 1 mg/mL. Cyclophosphamide was administered by oral gavage at a dose of 10 mg/kg bw/day and with an administration volume of 10 mL/kg bw.
On PND 56 ± 3 days, each animal of each group (C, LD, MD, HD and PC) was injected intravenously into the tail vein with 0.300 µg/kg of KLH as single dose (at a dose volume of 0.75 mL/kg bw). On this particular day, the oral gavage treatment with the vehicle, test item or cyclophosphamide, was performed after the i.v. treatment with KLH.
The immune response to KLH was evaluated by determining the titre of KLH-specific IgM antibody in the serum by ELISA, at the peak of the response before and after immunization (day 6).
Blood was sampled from all animals of this cohort by sublingual bleeding (under isoflurane anaesthesia) at least one week before immunization. On day 6 after immunization, blood was sampled for determination of KLH-specific IgM antibodies. The blood volume and tube, as well as parameters of serum preparation were documented in raw data.

- Splenic Lymphocyte Subpopulation and Organ Weights (Cohort 1A): 10 Male and 10 female Cohort 1A animals from each treatment group (1 male or 1 female per litter; randomly selected) were subjected for the investigation of pre- and post-natally-induced immunotoxic effects at termination. This included measuring the weight of lymph nodes (axillary lymph nodes), adrenal gland, thymus and spleen. Analysis of splenic lymphocyte subpopulation (lymphocytes, T-cells, CD4+ (helper T cells), CD8+ (cytotoxic T cells), B-lymphocytes, and natural killer (NK) cells) using one half of the spleen was performed.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving P males were subjected to necropsy after 10 weeks of dose administration.
- Maternal animals: All surviving P females were subjected to necropsy post-weaning (PND 21).

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The animals of F1-generation were subjected to necropsy depending on the scheduled ages for each cohort (Cohort 1A: week 13, 1B: week 20-25, Cohort 3: 8 weeks). Special attention was paid to the sexual organs of P and F1 animals for structural abnormalities.
- The F1 surplus pups after standardization and F1 pups not selected for cohorts (including runts) were sacrificed and subjected to gross necropsy on PND 4 and weaning (PND 22), respectively.
- F2 pups from Cohort 1B mating were sacrificed and subjected to necropsy at weaning (PND 22).
- Cohort 3 F1 animals were subjected only to gross necropsy and examined macroscopically for any structural abnormalities or pathological changes.

NECROPSY EXAMINATIONS
- Organ weights: performed for P and F1 (Cohort 1A) animals. Paired organs were weighed together. Organ weights of animals found dead or euthanised for animal welfare reasons were not taken. See table 1 for organs weighed for P and F1 animals at necropsy. The reproductive organs and organs identified as target organs in P and Cohort 1A animals were also weighed for Cohort 1B animals. See table 2 for organs weighed for Cohort 1B.
- Macroscopic examination of organs: Performed for P and F1 (Cohort 1A) animals. See table 1 for organs examined at gross necropsy.The reproductive organs and organs identified as target organs in P and Cohort 1A animals were also examined macroscopically for Cohort 1B animals. See table 2 for organs examined for Cohort 1B.
- Histopathology:
P animals: Performed for for all HD and control P animals. Histopathological examinations were extended to animals of the other dose groups, as treatment-related changes were observed in the HD group. Reproductive organs of all P animals that failed to mate, conceive, sire, or deliver healthy offspring, or for which oestrous cyclicity or sperm number, motility, or morphology were affected and all gross lesions were subjected to histopathological evaluation. See table 1 for tissues examined for P animals.
F1 (Cohort 1A) animals: Full histopathology of the organs listed in table 1 was performed on control and HD adult Cohort 1A animals. All litters were represented by at least 1 pup per sex. Histopathological examinations were extended to animals of the other dose groups, as treatment-related changes were observed in the HD group. All gross lesions from all dose groups were subjected to histopathological evaluation. Histopathology of lymph nodes (mesenteric and axillary) and bone marrow was performed on 10 males and 10 females Cohort 1A animals. Histopathology of thymus, spleen, and the adrenal glands was performed in all Cohort 1A animals. See table 1 for tissues examined for Cohort 1A animals.
F1 (Cohort 1B) animals: As the results from Cohort 1A examination there were no concearns for reproductive / endocrine toxicity and there were no triggers for further histopathology. Therefore, the examination was not extended to Cohort 1B animals.

Statistics:

A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed animals with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, thyroid hormones and foetal evaluation parameters including external, visceral, craniofacial and skeletal parameters were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. The statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 is considered as statistically significant).

Reproductive indices:

percent male mating index, female mating index, male fertility index, female fertility index, gestation index and live birth index

Offspring viability indices:

survival index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In terminally sacrificed Parental male and female animals, predominant clinical signs transiently observed in the majority of HD group animals were increased salivation (slight/moderate) and/or moving the bedding.
Low incidences of clinical signs including hairless area, crust, scratch/cut, eyelid closure, piloerection, chromodacryorrhea, corneal opacity and diarrhoea (single incidence), exophthalmos (left), wound, overgrown teeth and spontaneous reduced activity (slight) were observed in few animals on few days in all groups including controls.
As these findings showed no dose-dependency and were noted transiently, they were not considered to be toxicologically relevant.
The clinical signs salivation and moving the bedding in males and females were observed immediately after the dose administration and, therefore, were considered to be signs of a local reaction to the test item rather than a systemic adverse effect.

The weekly detailed clinical observations revealed no toxicologically relevant differences between the Parental treated and control groups during the entire study period. There were statistical significances observed in a few parameters in treated groups on a few occasions. However, observed statistically significant differences in a few parameters were either before initiation of treatment, not dose dependent/consistent or biologically relevant and, therefore, these findings were considered to be incidental and not related to the treatment with the test item.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item-related mortality was observed in males during the study period. In females, one LD animal (no. 138) was found dead on treatment day 6. The cause of death was due to gavage error during the dosing procedure.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In both male and female Parental animals, there was no test item-related effect observed on group mean body weight and mean body weight gain in the dose groups when compared with the controls. There were no statistically significant differences observed for these parameters between the dose groups and the control group, except for the following:
In males, statistically significantly lower body weight gain was seen between days 14-21 in LD and HD groups (46.49% and 37.84%, respectively) and between days 70-74 in LD and HD groups (135.23% and 204.55%, respectively) when compared to control. Statistically significantly increased body weight gain was found between days 49-56 in HD group (53.16%) males when compared to control. In females, statistically significantly higher mean body weight gain was observed between lactation days 14-21 (1.52%) in the HD group when compared to control. Due to the lack of dose dependency and consistency, these differences on Parental mean body weight gain and/or loss were not considered as test item-related and the mean body weight were found to be within the historical control range of this strain.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In correlation to body weight and body weight gain, food consumption in both males and females of the Parental generation tended to increase with the progress of the study in all groups.
No test item-related or statistically significant effect on food consumption was observed in males and females of the Parental generation during the whole study period except slight but statistically significantly higher group mean food consumption in Parental females between lactation days 0-4 in the MD group (24.77%) when compared to control. Due to the lack of dose dependency or consistency, this effect on food consumption was not considered to be adverse.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In 10 selected Parental males and females per group sacrificed at the end of the treatment period, no test item-related adverse effects were observed in haematological parameters in the dose groups when compared to the control group. In HD group males, moderately but statistically significantly higher mean WBC (35.1%) and marginally but statistically significantly higher mean RBC (5.4%), HGB (5.1%) and HCT (3.7%) were observed when compared to the control. In MD group males, higher mean HGB (6.0%) and HCT (4.9%) and lower RET percent (15.7%) were observed when compared to the control. In females, no statistical significance was observed when compared with the control. All these values were without any dose dependency or consistency; hence they were not considered to be adverse.
No test item-related effect was observed on coagulation parameters in Parental males and females when compared with the control, except for slight but statistically significantly higher mean PT value in HD group males (5.1%) and MD and HD group females (10.8-12.3%) when compared to the control.
All other group mean and most of the individual values for haematological parameters in male and females were comparable to the controls and within the normal range of variation; they were also within the range of historical control data of reproductive and developmental toxicity studies in this strain.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In 10 selected Parental males and females sacrificed at the end of the treatment period, marginal but statistically significantly lower creatinine (0.4%) was observed in HD group males when compared to the control; in HD group females, moderate but statistically significantly lower creatinine was observed in MD (30.4%) and HD (33.6%) groups when compared to the control. As the differences were marginal-to-moderate and all values were within the range of historical control data or without dose-dependency, this was not assumed to be toxicologically relevant.
All other group mean and most of the individual values for clinical chemistry parameters in male and females were comparable to the controls and within the normal range of variation.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The urinalysis performed in 10 selected males and females per group from Parental sacrificed at the end of treatment period revealed no test item-related effect in the dose groups when compared to control. All urinary parameters were in the normal range of variation.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

From the decedents in P-generation histopathological examination was available in one female from P-generation. Female No. 138 (P-generation, Group 2; found dead on Study Day 6): A limited set of organs and tissue was available for histopathology, which included the heart, lungs, esophagus and thymus. On the heart section, marked inflammation with tissue destruction, hemorrhage and granulation was found at/around the right atrium. There was also focal necrosis with hemorrhage in the esophagus. Thus, the cause of animal’s death was considered to be due to gavage error.
The most obvious finding in P-generation animals was diffuse urothelial hyperplasia in the urinary bladder, and this was observed in 20/25 males and 12/25 females of Group 3 and all males and females of Group 4. Diffuse urothelial hyperplasia was also found in the kidney of both sexes of Groups 3 and 4 and in the ureter of both sexes of Group 4, although it occurred at lower frequency or severity compared to that of the urinary bladder.
In the urinary bladder, the following findings that were considered to be treatment-related were also observed in both sexes of animals:
- Mixed inflammatory cell infiltration in the mucosa (epithelial layer and lamina propria): Groups 3 and 4;
- Increased incidence and/or severity of mononuclear cell focus/foci in lamina propria: Groups 3 and 4;
- Submucosal edema: Groups 3 and 4;
- Congestion: Group 4;
- Hemorrhage: one male of Group 3 and both sexes of Group 4.
In addition, the following changes recorded in the kidneys and ureters were also considered to be treatment-related.
Kidneys:
- Mixed inflammatory cell infiltration in the suburothelium: one male of Group 3 and in both sexes of Group 4;
- Fibrosis at/around the fornix and focal to multifocal interstitial fibrosis: males of Group 4;
There were also pelvic luminal precipitates, as well as appearance of foreign body gaint cells at/around the fornix, in males of Group 4;
- Increased incidence and/or severity of pelvic dilation, focal to multifocal mononuclear cell infiltration, tubular basophilia and tubular dilatation: males of Group 4;
Thus, the overall incidence and severity of the treatment-related renal lesions was higher in males than in females.
Ureters:
The ureters can be found by chance within or near the accessory sex organs, and in the present study, it was available in some sections from the prostate. Therefore, the observation of the ureters on the prostate sections was also made as a reference for histological evaluation.
- Increased incidence and severity of luminal dilatation: males of Group 4;
- Mixed inflammatory cell infiltration and submucosal edema were identified in the ureters on the prostate sections.
Small Intestine (Duodenum, Jejunum and Ileum)
Lipid accumulation in the lamina propria mucosa of the small intestine was observed in the HD animals from P-generation. This change was recognized histomorphologically as vacuolation or empty space of various sizes, most of which, especially larger vacuolation, were considered to be dilated lymphatic vessels, and some were within the cytoplasm of macrophages or present in the interstitium of the lamina propria.
Within the small intestinal segments, this lesion appeared predominantly in the jejunal region, and was not detected in the duodenum of females of P-generation.
The remaining microscopic findings recorded in this study were within the range of normal background lesions which may be observed in this study type and animals of this strain and age.
There was no test item-related histomorphological changes in the male and female reproductive organs in P-generation. In addition, no microscopic indicators for endocrine disruption were noted in the organs and tissues (including pituitary glands, adrenal glands and thyroid glands) examined in P-generation.
There were no statistically significant differences in all parameters including primordial follicles, growing follicles, the sum of primordial and growing follicles, antral follicles and corpora lutea.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

In Parental males and females (10/sex/group), group mean T4 and TSH levels in dose groups were comparable to control and there were no statistically significant changes observed.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The test item had no biologically or statistically significant effect on the oestrus cycle analysed during the 2 weeks pre-treatment and 2 weeks pre-mating period after the first administration in treatment groups when compared to the control. There were no considerable differences in the length or sequence of cycle stages between the treatment groups and the control group.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

The test item had no effect on epididymal sperm motility analysed from all males. Group mean motility values from Parental males of the dose groups were comparable to control.
Evaluation of sperm morphology from control and HD Parental males did not reveal any test item-related findings, except for a marginal but statistically significantly higher percent of abnormal sperm observed in the HD groups from Parental (8.42%) when compared to the control (6.91% in Parental). However, this was not considered to be adverse and these values were within the normal biological variation in this strain.
The test item had no effect on mean testis sperm count in Parental. Group mean sperm count from Parental males of dose groups were comparable to the control. The test item had no effect on mean testis weight in Parental males of dose groups when compared to the control.

Reproductive performance:

no effects observed

Description (incidence and severity):

None of the Parental females showed signs of abortion or premature delivery.
In Parental females, there were no test item-related or statistically significant effects on litter parameters including group mean total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, stillbirth, runt on PND 0 as well as number of alive pups, alive male and female pups and sex ratio on PNDs 4, 7, 14 and 21 when compared to the control. Statistical analysis of litter data revealed no significant effects in treatment groups when compared to the control.
There was no test item-related effect on pup mean weight, total litter weight, male and female litter weight on PNDs 0, 4, 7, 14 and 21 observed in Parental treatment groups when compared with the controls. There was no statistically significant change in dose groups compared to control.
There was no test item-related effect observed on the duration of pre-coital interval and the duration of gestation in the Parental female dose groups when compared to control and within the range of biological variation in treatment groups when compared to control.
There was no test item-related effect observed on the number of corpora lutea, implantation sites, alive pups on PND 0, percent pre-implantation loss and post- implantation loss in Parental treatment group females when compared with the corresponding control group.
There was no test item-related effect observed on the reproductive indices (percent male mating index, female mating index, male fertility index, female fertility index, gestation index and live birth index) in Parental dose group animals when compared to the respective control group.
The survival index of pups during PND 0 to 4, PND 4 (after interim sacrifice) to PND 14 and PND 14 (after interim sacrifice) to PND 21 in Parental females remained unaffected and within the range of biological variation in treatment groups when compared to the control.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Parental animals / reproductive toxicity

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects on reproductive function observed.

2

Key result

Dose descriptor:

NOAEL

Remarks:

Parental animals / general systemic toxicity

Effect level:

40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

bladder

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In terminally sacrificed males and females from various Cohort 1B clinical signs of increased salivation (slight/moderate) and/or moving the bedding were observed in the HD group.
The clinical signs salivation and moving the bedding in males and females were observed immediately after the dose administration and, therefore, were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect.
The weekly detailed clinical observations revealed no toxicologically relevant differences between the Cohort 1B treated and control groups during the entire study period. There were statistical significances observed in a few parameters in treated groups on a few occasions. However, observed statistically significant differences in a few parameters were either before initiation of treatment, not dose dependent/consistent or biologically relevant and, therefore, these findings were considered to be incidental and not related to the treatment with the test item.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Cohort 1B: One HD male (no. 430) died on day 25 by accidental incidence during the study. Two control females (nos. 442, 452, on days 28, 36, respectively), four LD females (nos. 469, 470, 476, 478 on days 22, 58, 21, 31, respectively) and one MD female (no. 497, on day 21) were found dead during the study period. At necropsy, no abnormalities were observed for animal nos. 469, 470, and 476. Animal no. 442 showed abnormal coloured (red) with fluid-filled lungs. Animal no. 452 showed lung adhesion with fluid-filled thoracic. Both animals did not show any clinical signs beforehand. Animal no. 478 showed advanced autolysis and lungs with fluid-filled (orange). Animal no. 497 showed abnormal coloured lungs, dark red.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1B: In both male and female Cohort 1B animals, there was no test item-related effect observed on group mean body weight and mean body weight gain in the dose groups when compared to control. Slight but statistically significantly higher group mean body weight was observed on days 11-29, 43, 64-71 and 84-98 in LD group (5.33-7.25%) males and on days 7-9 a slight but statistically significantly lower mean body weight (8.00-9.69%) was observed in HD group females when compared to control. Moderate but statistically significantly higher group mean body weight gain was observed between days 57-64 in LD group males (40.87%) and between days 119-126, moderate but statistically significantly lower mean body weight gain (84.96%) was observed in HD group males when compared to control. In females, moderate but statistically significantly higher mean body weight gain (85.03%) was observed on days 11-13 in MD group females when compared to control. Statistically significantly lower mean body weight (94.1% compared to control) was observed during lactation on day 21 in HD group females. Mean body weight gain was found to be lower between days 14-21 of lactation in all females, including controls.
Overall, mean body weight and mean body weight gain remained unaffected by the treatment with test item and values were in the normal range of variation throughout the treatment period when compared to the control group and they are within the range of historical control data of reproductive and developmental toxicity studies in this strain.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In correlation to body weight and body weight gain, food consumption in both males and females of the F1 Cohort 1B tended to increase with the progress of the study in all groups.
No test item-related or statistically significant effect on food consumption was observed in males and females during the whole study period except statistically significantly higher food consumption in LD group males (Cohort 1B) on days 1-7 (11.51%) and 63-70 (5.67%) when compared to control. Due to the lack of dose dependency or consistency, this effect on food consumption was not considered to be adverse.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Thickened wall of the urinary bladder was recorded in 13/20 males of Group 4 (HD) and in 1/20 females of Group 3 (MD) and 2/20 females of Group 4 (HD). In addition, dilatation of the ureter was recorded in 1/20 females of Group 4 (HD).
All other macroscopic findings recorded were lesions within the range of normal background changes which may be observed in rats of this strain and age, incidental appearances without corresponding histomorphological changes, or alteration representing normal physiology, and therefore, were deemed not to be related to treatment with the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

None of the Cohort 1B females showed signs of abortion or premature delivery.
Litter parameters of Cohort 1B females, including group mean total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, stillbirth, runt on PND 0, as well as number of live pups, male pups, number of female pups and sex ratio on PNDs 4, 7, 14 and 21 remained unaffected when compared with the controls. Statistical analysis of litter data revealed no significant effects in treatment groups when compared to control.
There was no test item-related effect on pup mean weight, total litter weight, male and female litter weight on PNDs 0, 4, 7, 14 and 21 observed in Cohort 1B treatment groups when compared with the controls. There was no statistically significant change in dose groups compared to control.
There was no test item-related effect observed on the duration of pre-coital interval and the duration of gestation in the Cohort 1B female dose groups when compared to control and within the range of biological variation in treatment groups when compared to control.
There was no test item-related effect observed on the number of corpora lutea, implantation sites, alive pups on PND 0, percent pre-implantation loss and post- implantation loss in Cohort 1B treatment group females when compared with the corresponding control group.
There was no test item-related effect observed on the reproductive indices (percent male mating index, female mating index, male fertility index, female fertility index, gestation index and live birth index) in Cohort 1B dose group animals when compared to the respective control group. The survival index of pups during PND 0 to 4, PND 4 (after interim sacrifice) to PND 14 and PND 14 to PND 21 in Cohort 1B females remained unaffected and within the range of biological variation in treatment groups when compared to the control.

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

P1 (Cohort 1B) / reproductive toxicity

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects on reproductive function observed

2

Key result

Dose descriptor:

NOAEL

Remarks:

P (Cohort 1B) / general systemic toxicity

Effect level:

40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

Target system / organ toxicity (P1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

bladder

ureter

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In terminally sacrificed males and females from various F1 Cohorts (1A and 1B), clinical signs of increased salivation (slight/moderate) and/or moving the bedding were observed in the HD group.
The clinical signs salivation and moving the bedding in males and females were observed immediately after the dose administration and, therefore, were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect.
The weekly detailed clinical observations revealed no toxicologically relevant differences between the F1 (Cohort 1A, 1B and 3) treated and control groups during the entire study period. There were statistical significances observed in a few parameters in treated groups on a few occasions. However, observed statistically significant differences in a few parameters were either before initiation of treatment, not dose dependent/consistent or biologically relevant and, therefore, these findings were considered to be incidental and not related to the treatment with the test item.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

F1 pup survival data: No test item-related effect on mean mortality of pups from PND 0 to 4, PND 4 (after interim sacrifice) to PND 14 and PND 14 (after interim sacrifice) to PND 21 in pups from Parental females was observed when compared to the respective control group.
Mean mortality of pups of the dose groups was comparable to the respective control and slight differences are considered as incidental and not related to treatment with the test item and they are within the range of historical control data of reproductive and developmental toxicity studies in this strain.
Adult F1 mortality data:
Cohort 1A: One LD female (no. 316) was found dead on day 22 and was cannibalized. One LD male (no. 239) and two females (no. 338, and no. 347) were moribund sacrificed for the animal welfare reasons on day 46 and on day 8 or 10, respectiively. Animal no. 338 showed abnormal and dark coloured lungs and animal no. 347 did not show any abnormalities at necropsy. Animal no. 239 showed wound/scar/crust, abdominal region of skin.
Cohort 3: No deaths.
The cause of the unscheduled deaths in Cohorts 1A and 1B was deemed to be due to accidental (e.g. gavage error) and not considered to be test item-related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A: In both male and female Cohort 1A animals, there was no test item-related effect observed on group mean body weight and mean body weight gain in the dose groups when compared with the control. Slight but statistically significantly higher group mean body weight was observed on days 1-3 (10.34-10.78%) and 7-15 (7.41-9.38%) in LD group males and on days 5-11 (6.26-6.89%) in LD group females when compared with the control. Slight but statistically significantly higher mean body weight gain was observed on days 3-5 in LD group females (15.5%) when compared to the control. However, in males, no statistical significance in mean body weight gain was observed between the groups at any time. As the observed differences on mean body weight and body weight gain in males and females were marginal and without dose dependence, they were not considered to be adverse.
Cohort 3: In both male and female Cohort 3 animals, there was no test item-related effect observed on group mean body weight and mean body weight gain in the dose groups when compared to control. Slight but statistically significantly higher mean body weight was observed in LD group males on days 9-11 and 22-36 (8.15-10.75%), in HD group males on day 29 (8.90), and in positive control group males on days 5, 9-11, and 22-29 (8.94-20.62%) when compared to control. Slight but statistically significantly lower mean body weight was observed in positive control group females on day 36 (10.04%) when compared to control. Slight but statistically significantly higher mean body weight gain was observed in LD group males on days 3-5 (79.1%) and days 1-36 (7.9%) when compared to control. In positive control group males, a statistically significantly lower mean body weight gain was observed on days 29-36 (60.7%) when compared to control. In females, a statistically significantly lower mean body weight gain was observed in the MD group on days 9-11 (37.7%) and in the positive control group on days 29-36 (48.6%) and days 1-36 (12.6%) when compared to control.
Overall in all F1 Cohorts, mean body weight and mean body weight gain remained unaffected by the treatment with test item and values were in the normal range of variation throughout the treatment period when compared to the control group and they are within the range of historical control data of reproductive and developmental toxicity studies in this strain

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In correlation to body weight and body weight gain, food consumption in both males and females of F1 cohorts (Cohort 1A and 3) tended to increase with the progress of the study in all groups.
No test item-related or statistically significant effect on food consumption was observed in males and females of the F1 cohorts during the whole study period except statistically significantly higher food consumption LD group females (Cohort 1A) on days 1-7 (10.77%) when compared to control. Due to the lack of dose dependency or consistency, this effect on food consumption was not considered to be adverse.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In 10 selected Cohort 1A males and females per group sacrificed at the end of the treatment period, marginally but statistically significantly higher mean RBC (8.4%) was observed in MD group males when compared with the control. Marginally but statistically significantly higher mean RBC (5.9%), HGB (7.0%) and HCT (6.1%) were observed in MD group females when compared with the control. As the differences were marginal and all values were within the range of historical control data, this was not assumed to be toxicologically relevant. All other group mean and most of the individual values for haematological parameters in male and females were comparable to the controls and within the normal range of variation.
In the absence of test item-related histopathological findings and effect on splenic lymphocyte subpopulation in the study, the above-mentioned increase or decrease in a few haematology parameters was not considered to be adverse.
No test item-related effect was observed on coagulation parameters in Cohort 1A males and females when compared with the respective control.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In 10 selected Cohort 1A males and females per group sacrificed at the end of treatment period, marginal but statistically significantly higher potassium (2.6%) in LD group males and lower total protein (6.0%) in HD group males were observed when compared to the control. In HD group females, moderate but statistically significantly lower ASAT (25.8%) and higher cholesterol (42.5%) were observed when compared to the control. In absence of test item-related histopathological findings and clinical signs, these effects in Cohort 1A animals were not considered to be toxicologically relevant.
All other group mean and most of the individual values for clinical biochemistry parameters in male and female Cohort 1A animals were comparable to the controls and within the normal range of variation.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The urinalysis performed in 10 selected males and females per group from Cohort 1A sacrificed at the end of treatment period revealed no test item-related effect in the dose groups when compared to control. All urinary parameters were in the normal range of variation.

Sexual maturation:

no effects observed

Description (incidence and severity):

All selected F1 male and female pups from all cohorts were checked daily for balano-preputial separation or vaginal patency, respectively, starting from PND 30 in males and PND 25 in females. No abnormalities of genital organs were observed in any of the pups.
Cohort 1A: No significant difference in group mean body weight and day of onset of vaginal opening in females and balano-preputial separation in males was observed in treatment groups when compared to control, except for a marginal but statistically significant reduction in vaginal opening observed in the LD group (PND 29.20) when compared to the control (PND 30.65) and this could be due to persistent diestrus of two female animals (nos. 309 and 311). It is not considered to be adverse and these values were within the normal biological variation in this strain.
Cohort 1B: No significant difference in group mean body weight and day of onset of the vaginal opening in females and balano-preputial separation in males was observed in treatment groups when compared to control.
Cohort 3: No statistically significant difference in group mean body weight and day of onset of the vaginal opening in females and balano-preputial separation in males was observed in treatment groups when compared to control, however marginal but statistically significant higher mean body weight on the day of balano-preputial separation was observed in the positive control (18.9%) and LD (15.7%) group when compared to the control. It is not considered to be adverse and these values were within the normal biological variation in this strain.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A: There were no statistically significant differences in the terminal body weights (TBW) in both sexes of F1-generation Cohort 1A.
In the organ weight determinations, the weight changes that could be related to treatment with the test item were found in the kidney of F1-generation Cohort 1A animals.
Kidneys: There was a statistically significant increase in relative (to TBW) weights in males of the HD group. In the HD group, there was also a tendency for an increase in the group mean values of absolute weights of males and of absolute and relative weights of females, although there were no statistically significant differences. These higher weight values were considered to be treatment-related.
The following statistically significant changes in the absolute and/or relative organ weights were recorded in females. However, there were no corresponding histological changes that could be related to the organ weight changes or no dose-response relationships were observed. Therefore, the weight differences recorded were considered to be incidental and not treatment-related.
- increase in absolute liver weights in the HD group;
- decrease in relative brain weights in the HD group;
- increases in absolute and relative spleen weights in the MD group.
Weights of spleen and thymus of Cohort 1A animals revealed no considerable changes that could indicate a test item-related immunotoxic effect.
F1 Pups not Selected for Cohorts: No test item-related effects on brain, spleen and thymus weights were observed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related gross external abnormalities of toxicological relevance on PND 0-20 were observed in the F1 pups of any of the groups from Parental females.
Cohort 1A adults: Test item-related gross findings were not recorded in F1-generation Cohort 1A animals.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

From the decedents in F1-generation Cohort 1A, histopathological examination was available in one male and one female from F1-generation Cohort 1A.
Male No. 239 (F1-generation Cohort 1A, Group 2; sacrificed moribund on Study Day 46): Grossly identified wound/scar/crust correlated microscopically with ulceration that arose on the surface of basal cell carcinoma of the skin, and this lesion was deemed to be the cause of animal’s morbidity. In this animal, enhanced hemopoiesis was found in the bone marrow (increased cellularity), spleen (massive extramedullary hematopoiesis) and liver (hemopoietic foci). This was considered to be secondary to the inflammatory response and possible loss of blood in the ulcerative lesion of the surface of the mass. In addition, there were stress-related reactions in the thymus (slight atrophy) and adrenal glands (minimal diffuse cortical hypertrophy).
Female No. 338 (F1-generation Cohort 1A, Group 3; sacrificed moribund on Study Day 8): Histopathological examination was available only for the lung sample. Only a minimum congestion was found in the lung sample, and the direct cause of animal's morbidity could not be established histologically. However, in the HD group of F1-generation, there was no unscheduled deaths likely to be due to the effects of the test item, and therefore, the morbidity in this animal was deemed not to be treatment-related.
The treatment-related findings qualitatively identical with that of P-generation animals were also observed in F1-generation Cohort 1A animals.
The most obvious finding was diffuse urothelial hyperplasia in the urinary bladder which was observed in 12/20 males and 4/19 females of Group 3 and all males and females of Group 4. This finding was recorded in the kidney of both sexes from Group 4 at a high frequency (all males and 16/19 females of Group 4), and it was also found in both sexes of Group 3 (two males and one female). Diffuse urothelial hyperplasia was also found in the ureter of both sexes of Group 4, at a lower frequency in comparison with the urinary bladder and kidneys.
Kidneys: The incidence and severity of some findings in the kidney were lesser in F1-generation Cohort 1A compared to that in P-generation, and these included mixed inflammatory cell infiltration in suburothelium, tubular dilatation and pelvic luminal precipitates. Further, there were no apparent differences in the incidence/severity of tubular basophilia and mononuclear cell infiltration between groups. Interstitial fibrosis, as well as fibrosis and foreign body giant cells at/around fornix, were not observed in the kidney of F1-generation Cohort 1A, although granuloma at/around fornix was found in one male of Group 4 instead.
Small Intestine (Duodenum, Jejunum and Ileum)
Lipid accumulation in the lamina propria mucosa of the small intestine was observed in the HD animals from F1-generation Cohort 1A. This change was recognized histomorphologically as vacuolation or empty space of various sizes, most of which, especially larger vacuolation, were considered to be dilated lymphatic vessels, and some were within the cytoplasm of macrophages or present in the interstitium of the lamina propria.
Within the small intestinal segments, this lesion appeared predominantly in the jejunal region, and was not detected in the duodenum and ileum of both sexes of F1-generation Cohort 1A.
The remaining microscopic findings recorded in this study were within the range of normal background lesions which may be observed in this study type and animals of this strain and age.
There was no test item-related histomorphological changes in the male and female reproductive organs in F1-generation Cohort 1A. In addition, no microscopic indicators for endocrine disruption were noted in the organs and tissues (including pituitary glands, adrenal glands and thyroid glands) examined in F1-generation Cohort 1A.
There were no statistically significant differences in all parameters including primordial follicles, growing follicles, the sum of primordial and growing follicles, antral follicles and corpora lutea.

Other effects:

no effects observed

Description (incidence and severity):

In Cohort 1A females, no biologically significant effect was observed on the duration of vaginal opening until first oestrus cycle in treatment groups when compared to control.
In this cohort, no biologically significant effect was observed on the oestrus cycle length or sequence of cycle stages between the treatment groups and the control group analysed from PND 75 for 2 weeks.

The test item had no effect on epididymal sperm motility analysed from all Cohort 1A males. Group mean motility values from Cohort 1A males of the dose groups were comparable to control.
Evaluation of sperm morphology from control and HD Cohort 1A males did not reveal any test item-related findings, except for a marginal but statistically significantly higher percent of abnormal sperm observed in the HD groups from Cohort 1A (8.23%) when compared to the control (5.84% in Cohort 1A). However, this was not considered to be adverse and these values were within the normal biological variation in this strain.
The test item had no effect on mean testis sperm count in Cohort 1A males. Group mean sperm count from Cohort 1A males of dose groups were comparable to the control. The test item had no effect on mean testis weight in PCohort 1A males of dose groups when compared to the control.

F1 pups on PND 4 and PND 21: In pups sacrificed on PND 4 (10/sex/group - pooled samples), T4 and TSH levels in treatment groups were comparable to the controls. In pups sacrificed on PND 21 (10/sex/group), T4 and TSH levels in treatment groups were comparable to the controls.
Cohort 1A adults: In males and females of Cohort 1A (10/sex/group), group mean T4 and TSH levels in treatment groups were comparable to the controls. Moreover, no test item-related effect of toxicological relevance was observed on thyroid weight and thyroid histopathology.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

Cohort 3: Control Group
Male: All males (10/10) showed an immune response after injection with KLH. It showed elevated anti-KLH specific IgM titers on day 6 after KLH injection, when compared to pre-levels (in average 2.8-fold higher). Animal no. 521 showed a 9.2-fold higher titer on day 6. Animal no. 528 did not show an elevated level of anti-KLH specific IgM titers on day 6 when compared to other animals in this group.
Female: All females (10/10) showed an immune response after injection with KLH. It showed increased anti-KLH specific IgM titers on day 6 when compared to pre-levels (in average a 5.1-fold higher). Animal no. 576 showed a 8.3-fold higher titer on day 6. Animal no. 577 showed a 13.6-fold higher titer on day 6 when compared to other animals in this group.
Cohort 3: Positive Control Group
Male: After immunization with KLH, decreased anti-KLH IgM titers (1.6-fold) were observed on day 6 in 4/10 male animals treated with the immunosuppressant cyclophosphamide when compared to pre-levels. Anti-KLH IgM titers of 10/10 animals were below the level of quantification (BLQ) or negative when compared to pre-levels. This showed the immune response to KLH in male animals treated with cyclophosphamide was effectively decreased.
Female: After immunization with KLH, decreased anti-KLH IgM titers (1.8-fold) were observed on day 6 in 7/10 female animals treated with the immunosuppressant cyclophosphamide when compared to pre-levels. Anti-KLH IgM titers of 9/10 animals were below the level of quantification (BLQ) or negative when compared to pre-levels. This showed the immune response to KLH in female animals treated with cyclophosphamide was effectively decreased.
Cohort 3: Dose Groups
Male: All male animals treated with test item (10/10 for each dose group) showed elevated anti-KLH specific IgM levels on day 6 after KLH-injection, when compared to pre-levels (on average 4.1-, 4.4- and 6.0-fold higher in the LD, MD and HD groups, respectively). This shows a clear immunological response in these male animals. Animal nos. 557 and 559 in the HD group showed 9.6-20.7-fold higher titer on day 6, when compared to other animals in this group. The slightly higher fold difference in the HD group could be due to these two individual animals with higher titer values.
Female: All female animals treated with test item (10/10 for each dose group) showed elevated anti-KLH specific IgM levels on day 6 after KLH-injection, when compared to pre-levels (on average 4.9-, 3.3- and 9.7-fold higher in the LD, MD and HD groups, respectively). This shows a clear immunological response in these female animals. Animal nos. 603, 605, 606, 607 and 610 in the HD group showed 9.9-77.7-fold higher titer on day 6, when compared to other animals in this group. The higher fold difference in the HD group could be due to these five individual animals with higher titer values.

Cohort 1A: Splenic Lymphocyte Subpopulation analysis
Analysis of spleen samples for splenic lymphocyte subpopulation (lymphocytes, T cells, CD4+ (helper T cells), CD8+ (cytotoxic T cells), B lymphocytes, and natural killer (NK) cells) from 10 male and 10 female animals per group from Cohort 1A revealed that there was a slight increase in mean lymphocytes and T cell populations in both males and females when compared to the control. There was no test item-related or statistically significant change in the number of splenic B cells. They were found to be comparable between all dose groups and control group in both males and females, except for a slightly higher mean splenic B cell value due to a high level of B cells in HD group female no. 263. Thus, there was no indication of an immunosuppressive effect of the test item on lymphocyte subpopulations.

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Generation:

F1 (cohort 1A)

Effect level:

40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Generation:

other: F1 (Cohort 1A) & F1 (Cohort 1B)

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects on reproductive organs observed

3

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

other: F1 (Cohort 1A) & F1 (Cohort 1B)

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse developmental effects observed

4

Key result

Dose descriptor:

NOAEL

Remarks:

developmental immunotoxicity

Generation:

F1 (cohort 3)

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse immunotoxic effects observed.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

bladder

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

F2 pup data: No test item-related effect on mean mortality of pups from PND 0 to 4, PND 4 to 21 in F2 pups from Cohort 1B female treatment groups was observed when compared to the respective control group.
Mean mortality of pups of the dose groups was comparable to the respective control and slight differences are considered as incidental and not related to treatment with the test item and they are within the range of historical control data of reproductive and developmental toxicity studies in this strain.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no test item-related effect on pup mean weight, total litter weight, male and female litter weight on PNDs 0, 4, 7, 14 and 21 observed in F2 from Cohort 1B treatment groups when compared with the controls. There was no statistically significant change in dose groups compared to control.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test item-related effects on brain, spleen and thymus weights were observed in F2 pups from Cohort 1B females.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related gross external abnormalities of toxicological relevance on PND 0-20 were observed in the F2 pups of any of the groups from Cohort 1B females.

Histopathological findings:

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:

developmental toxicity

Generation:

F2 (cohort 1B)

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse developmental effects observed.

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Concentration analysis of formulation samples was performed at three concentrations, 10.00 mg/mL, 25.00 mg/mL and 75.00 mg/mL in study weeks 1, 5, 9, 13 and in the last week of the study. The mean recoveries observed for the LD group were between 91.3% and 99.0% of the nominal value, between 91.8% and 97.1% of the nominal value for the MD group and between 88.5% and 96.3% of the nominal value for HD group. The mean recoveries observed in the LD, MD and HD groups were 95.8%, 94.5%, and 93.0% of the nominal concentration, respectively.

Nominal concentrations were confirmed for all dose groups, as mean measured concentrations were within acceptance criterion of 10%.

However, HD group sample no. 16 in week 13 did not meet this criterion with a recovery of 88.5%. It was decided that no reanalysis was necessary, since the mean value was still within the acceptance criteria.

Applicant's summary and conclusion

Conclusions:

In the extended one-generation reproductive toxicity study, conducted according to OECD Test Guideline 443 and in compliance with GLP, the NOAEL for general systemic toxicity for P and F1 (Cohort 1A) animals was concluded to be 40 mg/kg bw/day based on test substance related effects in the urinary system at 100 and 300 mg/kg bw/day; the reproductive toxicity NOAEL for P and F1 (Cohort 1A and 1B) animals was at least 300 mg/kg bw/day based on no adverse effects on male and female reproductive organs, on oestrous cycle and sperm parameters, and male and female fertility and performance; the developmental toxicity NOAEL for F1 (Cohort 1A and 1B) and F2 (Cohort 1B extension) was at least 300 mg/kg bw/day based on no adverse effects on pre- and post-natal development; the developmental immunotoxicity NOAEL for F1 (Cohort 3) was at least 300 mg/kg bw/day based on no adverse effects on immune system development and response.