Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Remarks:
Screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 July 2017 to
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-isopropyl-9H-thioxanthen-9-one
EC Number:
226-827-9
EC Name:
2-isopropyl-9H-thioxanthen-9-one
Cas Number:
5495-84-1
Molecular formula:
C16H14OS
IUPAC Name:
2-isopropyl-9H-thioxanthen-9-one
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Cream to pale yellow powder
- Storage conditions: Ambient temperature (10 - 30 °C)

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Upon arrival, males were 10 to 12 weeks of age, while females were 9 to 10 weeks of age. At the start of dosing males and females were considered sexually mature.
- Weight at study initiation: At the start of dosing, males weighed between 274.0 and 425.7 g; females weighed between 186.2 and 271.5 g.
- Fasting period before study: no
- Housing: During the pre-pairing phase, animals were housed in groups of up to four by sex and dose group. During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed. Following mating, females were housed individually during gestation and with their litter during the lactation phase. Males were returned to group-housing after the pairing phase. Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips or European Softwood bedding during the gestation and lactation phases.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: Animals were acclimatised for at least 3 weeks prior to initiation of dosing (males) or 5 days prior to smearing (females).

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Humidity: 30 – 70 %
- Air changes: 15 to 20 air changes/hour
- Photoperiod: 12 hours of light (fluorescent lighting) and 12 hours of dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Formulations were prepared daily for the first 16 days, while additional stability work was ongoing then weekly thereafter.
- The test material was formulated as a suspension in PEG 400 following dispensary SOPs. Method 8368780_O_01D at 5 mL/kg produced a formulation too viscous for dosing. Method 8368780_O_02D in combination with increasing dose volume to 8 mL/kg produced a formulation suitable for dosing.
- Formulations were stored at room temperature (15 to 25 °C) in a sealed container, protected from light.

VEHICLE
- Concentration in vehicle: 7.813, 12.5 and 200 mg/mL
- Amount of vehicle (if gavage): 8 mL/kg. Individual dose volumes were based on individual body weights.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
PROCEDURES
Linearity of Response
- A set of standard solutions over the approximate concentration range, 16.6 to 100.0 µg/mL, were analysed by ultra-performance liquid chromatography (UPLC). A calibration line was constructed for the calibration standard solutions. An injection of the lowest calibration standard, diluted by a factor of 100, was used to determine a limit of detection (LOD).

Precision and Accuracy
The precision and accuracy of the analytical procedure were determined as follows:
Sets of six standards, at concentrations of 16.66 and 99.97 µg/mL, were prepared from a stock solution.
A set of six standards were prepared at a concentration of 49.98 µg/mL, with the addition of 1 mL control vehicle (PEG 400) to 10 mL with acetonitrile.
A blank solution was also prepared with control vehicle, as mentioned previously.
A six-point calibration line was constructed from a different stock solution than the one mentioned previously.
All solutions were analysed by UPLC.
The concentration of validation standards sets were calculated from their UPLC responses using the calibration line.
The mean and standard deviations of these observations were used to determine the RSD and accuracy of the assay.
precision % = (standard deviation / mean) x 100
accuracy % = (mean calculated test material concentration/ theoretical test material concentration) x 100

Homogeneity and Stability
- Trial 1: Samples were removed in duplicate from the top, middle, and bottom of the 12.5 and 200 mg/mL formulations. Samples were analysed for test material concentration to determine homogeneity. Formulations were split into two aliquots. One aliquot was stored at room temperature (15 to 25 °C). Formulations were analysed as previously described to determine stability after 24 hours and 11 days. The second aliquot was stored refrigerated (2 to 8 °C) and was only analysed if degradation was observed in the room temperature formulations. These aliquots were not analysed.
- Trial 2: Samples were removed in duplicate from the top, middle, and bottom of the 7.813 mg/mL formulation. Samples were analysed for test material concentration to determine homogeneity. Formulation was split into two aliquots. One aliquot was stored at room temperature (15 to 25 °C). Formulation was analysed as previously described to determine stability after 24 hours and 10 days. The second aliquot was stored refrigerated (2 to 8 °C) and was only analysed if degradation was observed in the room temperature formulations. These aliquots were not analysed.

Achieved Concentration
Triplicate samples were removed from the test material formulations and were analysed. A single sample was taken from the control formulations and was analysed.

Analytical Procedure
The analytical procedure (Covance 8368780-01F) was used to determine homogeneity, stability and achieved concentration.

UPLC
Injection volume: 0.5 µL
Column: Acquity UPLC BEH C18 1.7 µm, 50 mm x 2.1 mm id
Temperature: 50 °C
Sample rack: 21 °C
Eluents: Eluent A: Water (500 mL, degassed before use), Eluent B: Acetonitrile (500 mL, degassed before use)
Flow rate: 0.6 mL/min
Nedle wash (Purge/ sample manager wash): Eluent B
Detector: Variable wavelength UV detector, wavelength 228 nm
Isocratic composition: 30 % Eluent A, 70 % Eluent B
Retention time: approximately 0.74 minutes
LOD: 0.1167 µg/mL (1 % of LLOQ)

Test material standard solution: 3-20 mg of test material was dissolved in acetonitrile to achieve a concentration of approximately 833 µg/mL. Sonication may be used. Two solutions (A and B) were prepared.

Generation of calibration line: Test material solution A (0.2, 0.6 and 1.0 mL) and test material solution B (0.4, 0.8 and 1.2 mL) are added to respective scintillation vials/ volumetric flasks and made to 10 mL with acetonitrile.

Extraction of samples: The formulations are diluted to within the range of the calibration line (approximately 16.6 to 100.0 µg/mL) using acetonitrile. Sonication may be used to aid dissolution. Control samples should, if possible, be diluted to an amount such that the analytical system equilibrium is not disturbed. If required vehicle can be injected undiluted but consideration should be given to the effects this will have on the analytical system. Control samples should not be diluted by an amount greater than that of the lowest sample concentration. The final sample and calibration samples are then submitted to UPLC.


RESULTS
Linearity of Response
A set of standard solutions was analysed by HPLC. The concentration/response curve was linear, with an intercept approaching zero. The correlation coefficient (r) was 0.9995. A minimum value of 0.9950 is considered acceptable; therefore, linearity of the response was considered acceptable.
The equation of the line was as follows: Y = 0.02313X – 0.02723
An injection at a concentration of 0.1667 µg/mL had a European pharmacopeia (EP) signal/noise ratio of 56.5 and was considered acceptable as the LOD.
The y-intercept should be ≤5.0 % of the response of the middle concentration. The y intercept was -2.51 % in the validation batch; therefore, it was considered acceptable.

Precision and Accuracy
Precision is expected to be less than 5 % RSD for this type of test, and accuracy is regarded as acceptable if it is between 90 and 110 %. The results were within these limits. The method was accepted in terms of precision and accuracy. No significant detector response was noted from the control solution, thereby confirming selectivity of the method.
16.66 µg/mL: Precision: 0.56 %, Accuracy: 104 %
49.98 µg/mL: Precision: 0.58 %, Accuracy: 102 %
99.97 µg/mL: Precision: 0.55 %, Accuracy: 101 %

Homogeneity and Stability
Formulations were considered homogeneous if the RSD of the results was ≤5.0 %. The results were within these criteria. Formulations were considered stable if the mean of the results at each time point were ±10 % of the mean at hour 0.
For trial 1 at 12.5 mg/L the mean % of nominal concentrations were 108, 102 and 99 % at 0 hours, 24 hours and 11 days, respectively. The relative standard deviations were 0.98, 0.33 and 0.35 % at 0 hours, 24 hours and 11 days, respectively.
For trial 1 at 200 mg/L the mean % of nominal concentrations were 102, 99 and 100 % at 0 hours, 24 hours and 10 days, respectively. The relative standard deviations were 1.66, 0.54 and 1.60 % at 0 hours, 24 hours and 10 days, respectively.
For trial 2 at 7.813 mg/L the mean % of nominal concentrations were 97, 101 and 97 % at 0 hours, 24 hours and 10 days, respectively. The relative standard deviations were 1.05, 1.37 and 2.57 % at 0 hours, 24 hours and 10 days, respectively.

Achieved Concentration
The mean percentage nominal concentration should be between 90 to 110 % with a relative standard deviation (RSD) ≤5.0 %. Results were within this range:
The mean % of nominal concentrations at the beginning of dosing were 98, 98 and 104 % in groups 2, 3 and 4, respectively. The relative standard deviations were 0.15, 0.83 and 0.77 % in groups 2, 3 and 4, respectively.
The mean % of nominal concentrations at the end of dosing were 103, 107 and 104 % in male groups 2, 3 and 4, respectively. The relative standard deviations were 1.18, 1.46, 0.71 % in male groups 2, 3 and 4, respectively. In female groups 2 and 3 the mean % of nominal concentrations at the end of dosing were 102 and 100 % with relative standard deviations 2.32 and 1.43 %, respectively.
Details on mating procedure:
- M/F ratio per cage: 1:1. During the pairing phase, one female was housed with one male from the same dose group.
- Length of cohabitation: Up to 10 days
- Proof of pregnancy: Mating was confirmed by the presence of either a vaginal plug in situ or sperm in a vaginal washing. Upon the confirmation of mating, vaginal washing was discontinued, and the male was removed from the cage. The day on which mating was confirmed was designated GD 0.
- Females without evidence of mating by Pairing Day 10 were paired for up to an additional 5 days with proven males of the same dose group.
Duration of treatment / exposure:
Males were dosed for 42 consecutive days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, and until the day before necropsy [post-pairing phase]).
Females were dosed for up to 56 days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, and until LD 13, inclusive, or Day 25 post-coitum for non-pregnant females). Some females were not dosed on LD 0 if they were observed to be starting, or had just completed, parturition.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The high-dose level of 1000 mg/kg/day was the limit dose recommended by the OECD 421 guideline. The dose level of 250 mg/kg/day was selected as an appropriate intermediate dose level. The low-dose level of 62.5 mg/kg/day was selected as the anticipated no observed adverse effect level (NOAEL).
- Rationale for animal assignment (if not random):Upon arrival, animals were assigned to dose groups using a total randomisation procedure. Animals were individually identified by electronic implant. Following the first full weighing (Day 1 of the predose period), group mean body weights and standard deviations were calculated and inspected to ensure no unacceptable differences occurred between groups. Replacements were made as required.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at the beginning and end of the working day for signs of ill health or overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed physical examination once daily from the start of dosing, including the day of necropsy. An individual record of the clinical condition of each animal was maintained.
- Animals were observed daily for the first 3 days of dosing; upon return to the home cage; and approximately 0.5, 1, 2, and 4 hours postdose. In the absence of any toxicologically significant postdose observations during the first 3 days of dosing, no further postdose observations were scheduled.

BODY WEIGHT: Yes
- Time schedule for examinations: Female body weights were recorded once during acclimation; on the first day of dosing; weekly prior to pairing; until confirmation of mating; on Gestation Day (GD) 0, 7, 14, and 20; and on LD 0, 1, 4, 7, 13 and 14 (terminal). From Study Day 24 (Pairing Day 9/ GD 3 through 8), body weights were recorded twice weekly, additional body weights were recorded on Pairing Day 10, and GD 4 and 17, where applicable.

FOOD CONSUMPTION:Yes
- The amount of food consumed was determined twice weekly from Day 1 of dosing (pre-dose on Day 1) and included the last day of the phase prior to pairing (both sexes). Daily food consumptions were recorded for females from GD 0 to 20 and LD 1 to 13.
- Consumption was calculated as g/animal/day.

WATER CONSUMPTION: No

NATURAL DELIVERY: Yes
- Animals were observed three times/day (at the beginning, middle, and end of each working day), starting when the first females reached GD 21 and until the last female had littered, or until a potential GD 25, whichever was earlier. Females were observed for signs of the start of parturition (for example, blood in the cage). The time and date of this observation were recorded, where possible, and marked the end of gestation; when not observed, the end of gestation was the day when the completion of parturition was recorded or on the day prior to when LD 1 observations were made. Abnormal observations of nesting, parturition, or nursing were recorded. It was considered important that the dam was disturbed as little as possible on LD 0. Any dead pups were removed and sent to necropsy to establish if they were born alive or dead (by looking for lung inflation).

SAMPLE COLLCECTION AND HANDLING
- Blood samples for thyroid hormone analysis (2 x 0.6 mL [serum separator tube], nominal) were withdrawn from the the tail vein (females) on the day of necropsy. Sampling was performed at a similar time on each occasion, and samples were collected by a controlled randomisation order where possible.
- Each blood sample was gently inverted several times, stored at room temperature and protected from light. Samples were centrifuged and aliquotted within 2 hours of blood sampling. The resultant serum was aliquotted into uniquely labelled clear polypropylene tubes and protected from light until frozen at <10°C.
- Total T4 = Thyroxine and TSH = Thyroid stimulating hormone tests were performed.

SACRIFICE
- Females were sacrificed by isoflurane anaesthesia on LD 14 (those that achieved pregnancy) or Day 26 post coitum (those that did not litter) after an overnight period without food. The Group 4 females remaining on 27 September 2017, (R0702 on LD 2, R0703 on LD 5, R0704 on GD 22, R0705 on LD 2 and R0706 on GD 14) were dispatched to necropsy and sacrificed early due to the mortality rate in the group and adversity of clinical signs observed. Once a suitable deep plane of anaesthesia was established, the major blood vessels were severed to exsanguinate the animal. Littered females were sacrificed in a controlled randomisation order, when possible. Females which did not litter or were not confirmed to have mated were sacrificed in animal order. Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded. The uterus of any apparently non pregnant female was immersed in a 10 % ammonium sulphide solution to reveal any evidence of implantation.

GROSS NECROPSY
- Organ weights were recorded at each scheduled sacrifice, excluding non-pregnant females: ovaries, thyroid with parathyroid and uterus with cervix. Paired organs were weighed together.
- The following tissues from each animal were retained in 10 % neutral buffered formalin, unless otherwise indicated: animal identification, gross lesions, mammary gland, ovaries, oviducts, pituitary, thyroid with parathyroid, uterus with cervix and vagina.

HISTOPATHOLOGY
- The following tissues were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with haematoxylin and eosin (for Group 1 (control), Group 3 (intermediate), Group 4 (high) and unscheduled deaths/sacrifices): gross lesions and ovaries. These tissues were all examined microscopically by the Study Pathologist.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- The uterus of any apparently non pregnant female was immersed in a 10 % ammonium sulphide solution to reveal any evidence of implantation.
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- On PND 4, litters were culled to 10 pups/litter, (five pups/sex when possible). Runts (pups considered unlikely to survive to weaning) were pre selected for cull. The remaining pups were selected randomly with preference to female pups. Culled pups underwent a macroscopic necropsy. Culling created a uniformly sized litter, which reduced differences in pup body weights due to litter size.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- On PND 1, 4, 7, and 13, litter size and pup sex were recorded.
- Pups were uniquely identified from PND 1.
- Daily records of mortality and changes in litter sizes were maintained. Where possible, pups found dead or in a moribund condition underwent a macroscopic necropsy.
- Each pup underwent a detailed clinical examination daily from PND 1.
- On PND 1, 4, 7, and 13 pup body weights were recorded.
- The ano-genital distance of all pups was recorded on PND 4.
- The number of nipples/areolae for male pups was counted on PND 13

GROSS EXAMINATION OF DEAD PUPS:
All animals which were sacrificed or died prior to their scheduled sacrifice were examined macroscopically, and all lesions were recorded.

SAMPLE COLLCECTION AND HANDLING
- Pups sacrificed on PND 13 had blood samples (2 x 0.6 mL [serum separator tube]) withdrawn for thyroid hormone analysis by cardiac puncture, to provide two samples for each litter (one sample from two males and one sample from two females, where possible).
- Samples were obtained from the litters which were unscheduled early terminal sacrifices. Each blood sample was gently inverted several times, stored at room temperature and protected from light. Samples were centrifuged and aliquotted within 2 hours of blood sampling. The resultant serum was aliquotted into uniquely labelled clear polypropylene tubes and protected from light until frozen at <10 °C.
Thyroid hormone test:
Total T4 = Thyroxine and TSH = Thyroid stimulating hormone

SACRIFICE
- Surplus pups culled on PND 4 (to standardise litter size) and pups sent to necropsy on PND 13 were sacrificed by an intraperitoneal injection of sodium pentobarbitone (overdose). Once a suitable deep plane of anaesthesia was established, major blood vessels were severed to exsanguinate the animal (decapitation for PND 4 pups). Food was not removed prior to scheduled necropsies.
- Surplus pups culled on PND 4 were discarded following blood sample collection. After sacrifice, external macroscopic observations were conducted for pups sacrificed on PND 13, paying particular attention to the external reproductive genitals. Full macroscopic examinations were conducted for all decedents; all lesions were recorded.
Statistics:
Data from test material-treated animals were compared with control data. Statistical analyses were performed, where appropriate.
Data for each sex were analysed separately, unless stated otherwise. Except when otherwise stated, tests were performed using a two-sided risk and were considered significant where P ≤ 0.05. By default, significant results were reported as *P ≤ 0.05, +P ≤ 0.01, and/or #P ≤ 0.001.
- Food Consumption (pre pairing phase [male and female] and post pairing [males]) - Procedure I (ANOVA) – Tox Reporting
- Thyroid hormones (male, female, and combined pups) Procedure I (ANOVA) – Tox Reporting
- Body weights (adult) - Procedure I (ANOVA) - Pristima
- Body weight gains (adult) - Procedure I (ANOVA) - Pristima
- Food consumption (gestation and lactation) - Procedure I (ANOVA) - Pristima
- Absolute organ weights and organ to terminal body weight ratios - Procedure I (ANOVA) - Pristima
- The mean number of oestrous cycles and mean cycle length - Procedure III
- Male and female mating, fecundity, and fertility indices - Procedure IV (one sided lower tail)
- The duration of gestation, number of implantation sites, number of pups born; number of pups alive on PND 1 and 4 (before culling), percentage male pups on PND 1, percent post-implantation loss, and live birth and survival indices PND 1 to 4 - Procedure III
- Pup weights (male, female, and combined) - Procedure II (litter size as the covariate)
- Ano-genital distance (males only) - Procedure II (cube root of mean pup weight as the covariate)
- Thyroid hormones (male, female, and combined pups) Procedure I (ANOVA) - Tox Reporting
Indices:
- Female mating index = (Mated females / Females cohabitated (excluding females sacrificed during cohabitation)) x 100
- Male mating index = (Number of males mating with at least 1 female / Number of males cohabitated with at least 1 female) x 100
- Female fecundity index = (Pregnant females / Mated females (excluding females with an undetermined pregnancy status)) x 100
- Male fecundity index = (Number of males impregnating at least 1 female / Number of males mating with at least 1 female) x 100
- Female fertility index = (Pregnant females / Females cohabitated (excluding females sacrificed during cohabitation or with an undetermined pregnancy status)) x 100
- Male fertility index = (Number of males impregnating at least 1 female / Number of males cohabitated with at least 1 female) x 100
- Mean duration of gestation (days) = Duration of gestation per female in group X / Number of dams in group X
- Mean number of implantation sites = Number of implantation sites in group X / Number of dams in group X
- Mean number of pups born = Number of pups born in group X /Number of dams in group X
- Mean number of pups alive PND 1 = Number of pups alive PND 1 in group X / Number of dams in group X
- % male pups PND 1 (litter) = (Number of male pups in litter on PND 1 / Number of pups in litter on PND 1) x 100
- % male pups PND 1 (mean) = Total % male pups on PND 1 in group X / Number of dams on PND 1 in group X
- Mean number of pups alive PND 4 before culling = Number of pups alive PND 4 before culling in group X /Number of dams in group X
- Mean number of pups culled PND 4 = Number of pups culled on PND 4 in group X / Number of dams in group X
- Mean number of pups alive PND 4 after culling = Number of pups alive PND 4 after culling in group X / Number of dams in group X
- Mean number of pups alive PND 7 = Number of pups alive PND 7 in group X / Number of dams in group X
- Mean number of pups alive PND 13 = Number of pups alive PND 13 in group X / Number of dams in group X

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Animals R0303, R0304, R0707, R0708 and R0710, administered 1000 mg/kg/day, were moribund sacrificed due to severity of clinical observations; these included thin appearance, soft/liquid/loose faeces, stained body and weight loss. Liquid/loose faeces and stained fur were also noted in found dead animals. Therefore, the clinical observations were considered test material-related.
- On occasion, from Pairing Day 1 onwards, incidences of soft and/or loose faeces and fur staining at various regions around the body for control and test material-treated in surviving animals were not consistently noted throughout the study. These observations were also noted along with brown staining around the anus/uro-genital region and wet fur. Though there was no dose response relationship, staining of fur in the top dose groups associated with weight loss and food consumption decreases, indicate general lack of grooming and suggest these may be indirectly attributed to test material administration.
- Salivation was noted across the groups, including control, but was not consistent or dose proportionate. Therefore, this finding was considered non-adverse.
- Incidences of sores/lesions and hair loss were noted as a general observation among all groups, including controls. These observations are noted as a background finding common in this age and strain of rat and are considered not test material related.
- Incidences of mouth rubbing were noted upon return to the home cage for animals administered 250 or 1000 mg/kg/day. This observation was related to the palatability of the test material and was considered non-adverse.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
- Several deaths attributed to the test material occurred.
- Three females administered 1000 mg/kg/day were sent to necropsy in a moribund condition: Animals R0707, R0708 and R0710 between Pre Pairing Day 13 and GD 3. Animals were removed from study due to a sudden marked reduction in food consumption with a correlating reduction in body weight (approximately 10 to 15 % body weight loss) and/or the severity of clinical observations. These observations included raised hair (piloerection), hunched posture, brown/yellow staining around the anus and/or ano-genital region, and abnormal soft/liquid faeces.
- Two females administered 1000 mg/kg/day had total litter loss, Animal R0709 was discovered to have only dead young at the completion of parturition and Animal R0701 on had lost all young by LD 2.
- The remaining females administered 1000 mg/kg/day were sacrificed early, just before or shortly after parturition, due to the number of unscheduled deaths in this group.
- Two females administered 250 mg/kg/day (Animals R0603 and R0606) were found dead on GD 22 and 23, respectively. These animals showed reduced body weight gain, fur staining, and abnormal soft faeces. Based on the in-life observations and as no evidence of dosing trauma or other cause of death was noted, the deaths of these animals were considered to be test material-related.
- One female administered 250 mg/kg/day (Animal R0795) had total litter loss on LD 1. The animal showed reduced body weight gains during gestation; however, the litter comprised of only one pup. No effect on food consumption or adverse clinical observations were recorded. While total litter loss was observed in the higher dose groups, incidences of total litter loss can be observed as a background finding in females with small initial litter sizes; therefore, this observation has an uncertain relationship to test material.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Females administered 1000 mg/kg/day showed a reduction in mean body weight gain toward the end of gestation (GD 14 and onwards). Individual females surviving to littering continued to show a reduction in body weight gain or body weight loss.
- No other effects on body weight were noted.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- An effect on mean food consumption was noted between Pre Pairing Day 1 and 8 (start of dosing) for females administered 250 or 1000 mg/kg/day. Females administered 1000 mg/kg/day were also noted to have reduced food consumption towards the end of gestation (GD 18 onwards).
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- At the terminal sacrifice, uterus:body weight ratio was reduced for females administered 250 mg/kg/day, (-10 %) compared with controls. Thyroid/parathyroid:body weight ratio was increased for females administered 250 mg/kg/day, (females: 15 %) respectively, compared with concurrent controls. Due to the lack of macroscopic findings, these were considered non-adverse.
- All other organ weight and organ weight ratio changes were attributed to normal biological variation and were considered not test material related as they were small in magnitude, not dose-dependent, inconsistent between sexes, due to normal inter animal variability, and/or lacked a microscopic correlate.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- No macroscopic findings that suggested test material-related effects were recorded in adult animals. Tissues were either macroscopically unremarkable or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age.
- A reduction in the total number of pups born was recorded for dams administered 250 or 1000 mg/kg/day (-27 and -48 %, respectively), which was due mainly to increased dam mortality at these dose levels. Pups were not examined from decedent or early terminal sacrifice dams sacrificed before parturition.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- No microscopic findings that suggested test material-related effects in the ovaries were recorded. Microscopic findings in these tissues were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain and age.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
- A reduction in T4 values (<41 vs. 53 nmol/L for control) and increased TSH values (0.43 vs. 0.32 µlU/L for control) for females administered 250 mg/kg/day were noted, however most individual data fell within background control ranges (T4= 30 to 58 nmol/L; TSH= 0.31 to 1.42 µlU/mL); no adverse test material-related effects was considered. Four of 10 females administered 1000 mg/kg/day were sampled for T4 and TSH. These values showed greater variation in results, but a general trend towards reduced T4 and increased TSH values was noted. While the small number of samples and the variation in sampling occasions should be taken into consideration, an effect of the test material could not be excluded.

Maternal developmental toxicity

Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
- A test material-related reduction in fertility index and pup survival was noted in animals administered 1000 mg/kg/day. The group administered 1000 mg/kg/day was sacrificed early due to the number of deaths, adverse toxicity in adult animals, and total litter loss.
- No effect was noted on other groups, including controls and any other test material treated groups.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
- Irregularity in the length of oestrous cycles was recorded for females R0702 and R0705 administered 1000 mg/kg/day, compared with controls during pre-dose phase. Animals R0704 and R0708 administered 1000 mg/kg/day showed increased oestrous length, compared with controls during pre-pairing phase. However, these females mated successfully and were pregnant. Therefore, this finding was considred to be of little biological relevance.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
62.5 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
changes in number of pregnant
clinical signs
food consumption and compound intake
mortality
organ weights and organ / body weight ratios

Maternal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: General toxicity: Mortality, body weight and weight gain.

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Body weight reduction was noted from PND 1 onwards for the surviving litters from dams administered 1000 mg/kg/day.
- No body weight effect was noted in other groups, including controls and any other test material-treated groups.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
- Effects on live birth index and/or litter survival were noted for the litters of dams administered 250 or 1000 mg/kg/day.
- Females administered 250 or 1000 mg/kg/day showed a reduction in live birth index, with higher rates of still births or pups not surviving to PND 1. These effects were considered test material-related.
- No effects were noted on other parturition or littering parameters
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
Females administered 1000 mg/kg/day also showed a reduction in survival index between PND 1 and 4. These effects were considered test material-related.
- All pups from one dam administered 1000 mg/kg/day (Animal R0701) were found dead with no milk in the stomach, and all pups from another dam administered 1000 mg/kg/day (Animal R0709) were stillborn. No macroscopic findings were recorded for any pups in the litters from the other three dams administered 1000 mg/kg/day (Animals R0702, R0703, and R0705) to suggest test material related effects.
External malformations:
no effects observed
Description (incidence and severity):
- No macroscopic findings were recorded for any pups examined from groups administered 62.5 or 250 mg/kg/day, with the exception of one decedent female administered 250 mg/kg/day (Animal R0795) that had only one pup. The relationship of this reduction in reproductive performance to the test material was uncertain.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Ano-Genital Distance: No effect of the test material was observed in the litters surviving to assessment at PND 4 from dams administered 62.5 or 250 mg/kg/day. Only one litter from dams administered 1000 mg/kg/day survived to the age of assessment.
- Nipple/Areolae Count: No incidence of nipple/areolae were noted for any male pup from litters surviving to PND 13. No litters from animals administered 1000 mg/kg/day survived to PND 13; as such, assessment was made on litters from controls and animals administered 62.5 or 250 mg/kg/day only.
- Thyroid hormone values were similar between all groups, including controls. Pup thyroid hormone were considered unaffected by the test material.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
62.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: Decreased live birth, litter survival consequent to maternal toxicity.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table 1: Summary of Parturition and Litter Data and Pup Survival

Measurement

Group 1

Group 2

Group 3

Group 4

Statistics

Duration of gestation (days)

Mean

23.2

23.1

22.9

23.5

P3

SD

0.42

0.33

0.83

1.00

N

10

9

8

4

Number of implantation sites

Mean

11.6

12.6

10.1

12.3

P3

SD

3.06

1.81

4.32

1.26

N

10

9

8

4

Number of pups born

Mean

10.3

11.2

9.4

11.3

P3

SD

2.21

1.99

3.70

1.50

N

10

9

8

4

Number of pups alive PND1

Mean

10.3

11.2

9.3

10.0

P3

SD

2.21

1.99

4.03

2.45

N

10

9

8

4

% male pups PND 1

Mean

48.2

41.9

51.2

57.2

P3

SD

10.13

13.63

16.49

5.12

N

10

9

7

4

Number of pups alive PND 4 before culling

Mean

10.3

11.2

10.6

3.3

P3

SD

2.21

1.99

1.62

6.50

N

10

9

7

4

Number of pups culled PND 4

Mean

1.0

1.6

1.0

3.0

X

SD

1.25

1.33

1.00

-

N

10

9

7

1

Number of pups alive PND 4 after culling

Mean

9.3

9.7

9.6

10.0

X

SD

1.34

1.00

0.79

1

N

10

9

7

1

Number of pups alive PND 7

Mean

9.3

9.7

9.6

0.0

X

SD

1.34

1.00

0.79

-

N

10

9

7

1

Number of pups alive PND 13

Mean

9.3

9.7

9.6

-

X

SD

1.34

1.00

0.79

-

N

10

9

7

0

Post-implantation survival index %

Mean

91.0

89.3

94.4

91.8

P3

SD

13.79

10.07

7.11

6.85

N

10

9

8

4

Live birth index %

Mean

100.0

100.0

87.5

89.3

P3

SD

0.00

0.00

35.36

20.83

N

10

9

8

4

Survival index PND 1-4 %

Mean

100.0

100.0

100.0

25.0+r

P3

SD

0.00

0.00

0.00

50.00

N

10

9

7

4

Survival index PND 4-7 %

Mean

100.0

100.0

100.0

0.0

X

SD

0.00

0.00

0.00

-

N

10

9

7

1

Survival index PND 7-13 %

Mean

100.0

100.0

100.0

-

X

SD

0.00

0.00

0.00

-

N

10

9

7

0

PND = Postnatal Day

P3 = Kruskal-Wallis and Wilcoxon

*r = Wilcoxon Rank Sum Test Significant at 0.05 level

+r = Wilcoxon Rank Sum Test Significant at 0.01 level

#r = Wilcoxon Rank Sum Test Significant at 0.001 level

X = not analysed

DISCUSSION

Daily oral (gavage) administration of 250 or 1000 mg/kg/day test material was not well tolerated by adult animals; this had a subsequent effect on pregnancy and lactation in animals administered 1000 mg/kg/day.

Seven decedents attributed to the test material were noted; five animals administered 1000 mg/kg/day (three female and two male) and two females administered 250 mg/kg/day. These animals were observed to have a reduction in food consumption, weight loss, and adverse clinical observations.

A reduction in body weight/body weight gain was observed for animals administered 250 or 1000 mg/kg/day. Animals administered 250 mg/kg/day, excluding decedent animals, generally showed recovery after the first week of dosing. Animals administered 1000 mg/kg/day were observed to have body weight effects at other later points during the study. Food consumption was also, on occasion, noted to be reduced for females administered 1000 mg/kg/day. These effects on food consumption and body weight were attributed to the test material.

A reduction in thyroxine (T4) values were observed for males administered 1000 mg/kg/day. These changes were considered test material-related.

At the terminal sacrifice, uterus:body weight ratio was reduced for females administered 250 mg/kg/day, compared with controls. Seminal vesicle:body weight was reduced for males administered 62.5, 250, or 1000 mg/kg/day and prostate:body weight ratio was reduced for males administered 250 or 1000 mg/kg/day. Thyroid/parathyroid:body weight ratio was increased for males administered 62.5, 250, or 1000 mg/kg/day and females administered 250 mg/kg/day. No macroscopic or microscopic findings that suggested test material-related effects in the testis, epididymis, or ovary were recorded.

Animals administered 250 or 1000 mg/kg/day showed test material-related effects in some littering parameters, including live birth, litter survival, and/or PND 1 to 4 survival index. These effects were below our background data ranges. Pups from dams administered 1000 mg/kg/day were also noted to have reduced body weights and adverse clinical observations, including cold to touch, pale, and no milk in stomach; these findings indicated a lack of maternal care post littering. These observations were considered test material related and are adverse.

No other pup developmental effects were noted.

No effect on gonadal function, mating behavior, conception, development of the conceptus, parturition, or offspring growth until LD 13 was noted; the increased mortality in litters from animals administered 250 or 1000 mg/kg/day may, therefore, have been attributed to adverse maternal toxicity, but the exact cause was uncertain.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, based on the adverse findings noted, the no observed adverse effect level (NOAEL) for systemic, and the reproductive and developmental toxicity is 62.5 mg/kg/day.
Executive summary:

The developmental toxicity of the test material was investigated in a Reproduction/Development Toxicity Screening study in rats, conducted in accordance with the standardised guideline OECD 421, under GLP conditions.

The objective of the study was to screen for effects of the test material on male and female reproductive performance (i.e. gonadal function, mating behaviour, conception, development of the conceptus and parturition) and offspring growth until Lactation Day (LD) 13.

Four groups of 10 animals/sex/group were administered 0 (control material [vehicle]), 62.5, 250, or 1000 mg/kg/day test material orally by gavage at a volume of 8 mL/kg. The control material (vehicle) was PEG 400. The test material was administered to males for 42 days (pre-pairing, pairing, and post-pairing) or to females for up to 56 days (pre-pairing, pairing, gestation, and up to LD 13).

Assessment of toxicity in adults was based on clinical observations, body weights, food consumption, oestrous cycles, mating, fertility and pregnancy indices, and offspring parameters. Pup clinical observations, litter size, sex, and body weights were recorded. Ano-genital distance was recorded on Postnatal Day (PND) 4, and nipple retention was recorded for male pups on PND 13. One pup/sex/litter/dose group was selected; these pups had their thyroid preserved.

Complete necropsies were performed on all animals, and any macroscopic abnormalities were noted. Blood samples for thyroid hormone assessment were collected at necropsy from all adult animals and selected pups on PND 4 and 13. Organ weights and microscopic examinations were conducted as indicated.

Daily oral (gavage) administration of 250 or 1000 mg/kg/day test material was not well tolerated by adult animals; this had a subsequent effect on pregnancy and lactation.

Seven test material-related decedents were noted; five animals administered 1000 mg/kg/day and two animals administered 250 mg/kg/day. These animals were observed to have a reduction in food consumption, weight loss, and adverse clinical observations. Additionally, two additional females administered 1000 mg/kg/day and one female administered 250 mg/kg/day were sacrificed after they lost their litters, between Lactation Day (LD) 1 and 2. Due to the number of decedents on study the five remaining females administered 1000 mg/kg/day were sacrificed early, just before or shortly after parturition, due to the severity of the clinical observations.

Staining of fur in animals administered 250 or 1000 mg/kg/day associated with weight loss and food consumption decreases, indicate general lack of grooming and suggest these to be indirectly related to test material administration.

An initial reduction in body weight gain was noted in males administered 250 mg/kg/day, males administered 1000 mg/kg/day show reduced body weight gain throughout the entire study duration. In males administered 1000 mg/kg/day, reduction in thyroxine (T4) values were also observed. These effects were attributed to the test material.

A reduction in mean food consumption was noted during the first week of dosing for females administered 250 or 1000 mg/kg/day and from GD 18 onwards for females administered 1000 mg/kg/day. Females administered 1000 mg/kg/day showed a reduction in mean body weight gain from GD 14 and onwards and those females surviving to littering continued to show a reduction in body weight gain or body weight loss. There was also an indication of increased length of oestrous cycles for females administered 250 or 1000 mg/kg/day, but this was not associated with effects on fertility.

Uterus, seminal vesicle and prostate weight were reduced, and the thyroid weight increased in the test material treated animals compared to control. Due to the lack of macroscopic findings, these changes were considered non-adverse.

No microscopic findings that suggested test material-related effects in the testis, epididymis, or ovary were recorded.

Due to high number of decedent animals at 1000 mg/kg/day a conclusive assessment of littering and pup survival parameters was not possible. Animals administered 250 mg/kg/day showed test material-related effects on live birth, litter survival, and/or PND 1 to 4 survival index. Increased mortality was also noted in litters from animals administered 250 mg/kg/day. No other pup developmental effect was noted.

No effect on gonadal function, mating behaviour, conception, development of the conceptus, parturition, or offspring growth until LD 13 was noted.

Under the conditions of this study, based on the adverse findings noted, the no observed adverse effect level (NOAEL) for systemic, and the reproductive and developmental toxicity is 62.5 mg/kg/day.