Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Qualifier:
according to
Guideline:
other: • OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test,
Version / remarks:
July 2000.
Qualifier:
equivalent or similar to
Guideline:
other: OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test,
Version / remarks:
July 2000
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
white to brownish
Details on test material:
Batch 151222

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han) (outbred, SPF-Quality).
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age/weight at study initiation: At initiation of dosing, males were 10 weeks old and weighed between 263 and 312 g and females were 13 weeks old and weighed between 189 and 234 g.
- Fasting period before study: no
- Housing:
On arrival and following the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not be left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
Sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet (e.g. ad libitum):
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
- Water (e.g. ad libitum):
Free access to tap-water. During motor activity measurements, animals did not have access to water for a maximum of 2 hours.
- Acclimation period:
At least 5 days prior to start of pretest (females) or treatment (males).

DETAILS OF FOOD AND WATER QUALITY:
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: dosing was initiated on 03 May 2017. To: The in-life phase of the study was completed on 05 Jul 2017.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared within 8 days prior to dosing and were homogenized to a visually acceptable level. The formulations were filled out in daily proportions and kept in the refrigerator in the dark until needed. Adjustment was made for the specific gravity of the vehicle. No correction was made for the purity/composition of the test item.

- VEHICLE
- Justification for use and choice of vehicle (if other than water):
The choice of vehicle was based on trial formulations performed at Charles River Den Bosch.
- Concentration in vehicle:
15, 50 and 150 mg/kg
- Amount of vehicle (if gavage):
5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
- Any other deviations from standard protocol:
/
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis were conducted on a single occasion during the treatment phase according to a validated method (Test Facility Study No. 516073, ABL No. 16303).

Immediately after sampling, samples were stored on dry ice. Samples remained on dry ice until receipt at ABL, The Netherlands.
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

Concentration Analysis
Duplicate middle samples for Groups 1 and 3 and duplicate top, middle, and bottom samples for Groups 2 and 4 (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration.
Homogeneity Analysis
Duplicate top, middle, and bottom samples for Groups 2 and 4 (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
Stability Analysis
Stability analyses performed previously in conjunction with the method development and validation study (ABL No. 17053) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for ABL No. ABL17053. In this study, stability for at least 5 hours at room temperature, 8 days in the refrigerator and 3 weeks in the freezer was confirmed over the concentration range 1 to 200 mg/mL.
Duration of treatment / exposure:
Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females that delivered were treated for 50-56 days (most females) or 62 days (one female), i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for 39, 41 or 43 days.

Routinely, females that are littering are left undisturbed. In this study, female nos. 48 (Group 1), 56, 59 (Group 2), and 64 (Group 3) were not dosed on one occasion as they were littering at the time of dosing. The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of an acute oral toxicity study (Test Facility Study No. 515451; treatment-related mortality at 300 mg/kg) and a dose range finder (Test Facility Study No. 515454)
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
Mortality
F0: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.
F1: The numbers of live and dead pups were determined on PND 1 and daily thereafter. If possible, defects or cause of death were evaluated.
Clinical observations
F0: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals, at least 1 hour (± 15 min) after dosing (on the peak period of anticipated effects after treatment).
The time of onset, grade and duration of any observed sign was recorded.
F1: Clinical observations were performed at least once daily for all pups.
Cohabitation/mating
After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
General reproduction data
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day. Palpation was used to aid in confirmation of pregnancy. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females were examined for evidence of abortion or premature delivery. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.


DETAILED CLINICAL OBSERVATIONS: Yes
Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
F0: Males and females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.
F1: Live pups were weighed on PND 1, 4, 7 and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes
Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: /

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: /
Postmortem examinations (parental animals):
SACRIFICE
All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.
Scheduled necropsies were conducted on the following days:
Males: Following completion of the mating period (a minimum of 28 days of administration).
Females which delivered: PND 14-16.
Females which failed to deliver: Post-coitum Days 25 or 26 (females with evidence of mating)
Female with total litter loss: Within 24 hours of litter loss.

GROSS NECROPSY
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.
Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
The number of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites (Salewski staining prepared at Charles River Den Bosch using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:
Selected 5 animals/sex/group (see Allocation):
Adrenal glands
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries
Prostate
Seminal vesicles, including coagulating glands
Spleen
Testes
Thymus
Thyroid, including parathyroid if detectable
Uterus, including cervix
All remaining animals:
Epididymides
Prostate
Seminal vesicles, including coagulating glands
Testes
Thyroid, including parathyroid if detectable


The tissues indicated in Table 1 were prepared for microscopic examination.
Postmortem examinations (offspring):
SACRIFICE
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. A
On PND 13-15, from 2 pups per litter (if possible from one male and one female) blood samples were collected at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands), between 7.00 and 10.30 a.m., followed by exsanguination. Blood was collected into serum tubes.
All remaining pups (PND 13-15) were sacrificed using Euthasol® 20% (AST Farma B.V., Oudewater, The Netherlands) by intraperitoneal (ip) injection.

GROSS NECROPSY:
All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood sampling, was preserved in 10% buffered formalin.
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

HISTOPATHOLOGY / ORGAN WEIGTHS: /
Statistics:
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test (was applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test as applied to motor activity data to determine intergroup differences.
Reproductive indices:
Mating (%)
Precoital time
Fertility index (%)
Gestation index (%)
Duration of gestation


Offspring viability indices:
Post-implantation survival index (%)
Live birth index (%)
Percentage live males at First Litter Check (%)
Percentage live females at First Litter Check (%)
Viability index (%)
Lactation index (%)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Salivation seen after dosing for all animals at 150 mg/kg (up to moderate degree) and incidentally across the other groups (slight degree) was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing).

Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
One female at 50 mg/kg (no. 69) was sacrificed on Day 1 of lactation due to total litter loss
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights of treated males and females remained in the same range as controls over the entire treatment period.
Statistically significantly lower mean body weight gain was noted in females at 150 mg/kg on Day 13 of the lactation period. As mean body weights were similar as controls on Day 13 of lactation, the slightly lower body weight gain was not considered to be toxicologically relevant.
Statistically significantly higher mean body weight gain was noted in treated males at the end of the treatment period, i.e. Day 15 of the mating period. As the differences from controls showed no dose-related trend, they were considered to be unrelated to treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in food consumption were noted by treatment up to 150 mg/kg.
In females treated at 150 mg/kg, mean food consumption (before and after allowance for body weight) was slightly higher during the pre-mating period. As the changes were only minimal, not statistically significant and remained within the range considered normal for rats of this strain and age, these changes were not considered to be toxicologically relevant.
In males, food consumption before or after allowance for body weight was similar between treated and control animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Haematological parameters were considered not to be affected by treatment.
Isolated statistically significant variations noted (higher platelets in males at 15 mg/kg and lower mean corpuscular volume (MCV) in females at 50 mg/kg) were considered unrelated to treatment due to the absence of a dose-related response.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males, a dose-related increased mean concentration of bile acids was noted, which was statistically significant at 150 mg/kg (relative difference of 88% compared to controls). This was considered to be partly caused by one male (no. 34) showing a notably high value of 56.5 µmol/L, that was above the P95 value of available historical control data. Other individual bile acid values of treated males remained in the range considered normal for rats of this age and strain.
The other statistically significant difference noted in clinical biochemistry parameters (i.e. lower urea in females at 15 mg/kg) was considered to be unrelated to treatment due to the absence of a dose-related response.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Functional observation parameters were considered not to be affected by treatment.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment.
Motor activity data showed a similar habituation profile with a decreasing trend in activity over the duration of the test period for all groups.
Findings of note were lower mean values for total movements and ambulations in males at 150 mg/kg (relative differences from controls: 28 and 19%, respectively) and higher values in females at 150 mg/kg (relative differences from controls: 43 and 74%, respectively). Statistical significance was not achieved, there were no corroborative clinical signs or changes in other parameters in the neuromuscular domain (including gait, air righting reflex and grip strength), and all values at 150 mg/kg remained within the range of available historical control data. In combination with the inconsistent direction of the differences in males (lower values) and females (higher values), these differences in motor activity were considered not to represent a test item-related effect.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations.
The recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item‑related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations.
The recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item‑related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Other effects:
no effects observed
Description (incidence and severity):
No effect on T4 serum levels or blood coagulation observed.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment.
Most females had regular cycles of 4-5 days. Extended di-estrus during the mating period occurred in two females (no. 52 at 15 mg/kg and no. 62 at 50 mg/kg), and two females had an irregular cycle during the pre-mating period (no. 42 of the control group and no. 55 at 15 mg/kg). These females all had a normal litter with living pups. These incidental findings were considered to be unrelated to treatment as they occurred in absence of a dose-related trend.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
The following couples had no offspring: 2/10 control group couples (female/male no. 41/1: not pregnant; female/male no. 47/7: no evidence of mating) and 1/10 couples at 100 mg/kg (female/male no. 60/20: not pregnant). Histopathology did not reveal any changes in the reproductive organs that could explain this.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Mating index was not affected by treatment. All females showed evidence of mating.
Precoital time was not affected by treatment.
Number of implantation sites was considered not to be affected by treatment.
Compared to the control group, the mean number of implantation sites was lower at 50 mg/kg and 150 mg/kg (10.1 and 11.2 versus 13.6). These lower group means could be explained by low numbers of implantation sites in a few females (nos. 67 and 69 at 50 mg/kg and nos. 75 and 78 at 150 mg/kg with 5, 1, 5 and 5 implantation sites, respectively). Such low numbers of implantation sites occasionally occur in untreated controls. Moreover, the differences from the control group showed no dose-related trend and were not statistically significant. Therefore, this finding was considered unrelated to treatment and not toxicologically relevant.

Fertility index was not affected by treatment.
Except for one control female (no. 46), one female at 50 mg/kg (no. 70) and one female at 150 mg/kg (no. 74), all mated females were pregnant. These cases of non-pregnancy, all without related histopathology changes in reproductive organs, were considered to be unrelated to treatment as the incidence showed no dose-related trend.

Gestation index and duration of gestation were not affected by treatment. The gestation index was 78% for the 50 mg/kg group and 100% for the other groups.
Except for two females at 50 mg/kg (no. 67 with implantation sites only and no. 69 with one dead pup only at first litter check), all pregnant females had live offspring. These cases of failed pregnancy without related histopathology changes in reproductive organs were considered to be unrelated to treatment as the incidence showed no dose-related trend.
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
The total number of offspring born compared to the total number of uterine implantation sites was not affected by treatment. The post-implantation survival indices were 88, 89, 82 and 88% in Groups 1, 2, 3 and 4, respectively.
For female nos. 72 and 73 (150 mg/kg), the number of pups born was slightly higher than the number of implantation sites. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation.

Litter size was considered not to be affected by treatment.
Compared to the control group, the mean number of living pups at first litter check was lower at 50 and 150 mg/kg (9.1 and 9.6 versus 11.9). These lower group means could be explained by low numbers of living pups in a few litters (no. 69 at 50 mg/kg with no living pups, and nos. 75, 78 and 79 at 150 mg/kg with 3 to 5 living pups), which was related to the low number of implantation sites in some of these females. Such low numbers of living pups occasionally occur in untreated controls. Moreover, the differences from the control group showed no dose-related trend and were not statistically significant. Therefore, this finding was considered unrelated to treatment and not toxicologically relevant.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
> 150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
neuropathology
histopathology: non-neoplastic
histopathology: neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment.
The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of live offspring on Day 4 before culling compared to the number of live offspring on Day 1 after littering was not affected by treatment. The viability indices were 95, 100, 97 and 99% in Groups 1, 2, 3 and 4, respectively.
Five pups of the control group (of litter nos. 42, 44, 48 and 49), two pups at 50 mg/kg (litter nos. 63 and 64), and one pups at 150 mg/kg (litter no. 72) went missing at Days 2-4 of lactation and were most likely cannibalized. This post-natal loss was considered to be unrelated to treatment as the incidence showed no dose-related trend and remained within the range considered normal for pups of this age.

The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment. The lactation indices were 100, 96, 100 and 93% in Groups 1, 2, 3 and 4, respectively.
Mortality between Days 5-13 was limited to one litter at 15 mg/kg and one litter at 150 mg/kg. Three pups of litter no. 58 (15 mg/kg) went missing on Days 5-9 and four pups of litter no. 72 (150 mg/kg) went missing or were found dead on Days 6-9. Pups missing were most likely cannibalised. At 150 mg/kg, the mortality incidence differed statistically significantly from that in the control group in which no pups went missing or died after PND 4. This incidental pup mortality was considered to be unrelated to treatment as it affected only one litter in each group and as the incidence showed no dose-related trend and remained within the range considered normal for pups of this age.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were considered not to be adversely affected by treatment.
Compared to controls, mean body weights of male and female pups at 150 mg/kg were slightly higher from birth until PND 13. The differences, ranging from 6% at PND 1 to 9% at PND 13, were not statistically significant. Moreover, lower rather than higher pup body weights are regarded as indicator of developmental toxicity. For these reasons, the slightly higher pup weights at 150 mg/kg were considered not to represent an adverse effect of the test item.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 13-15 pups were not affected by treatment.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of the macroscopic findings noted incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Sex ratio was not affected by treatment.

Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment.
In the absence of a dose-related trend, the lower mean normalized anogenital distances noted in female pups at 15 and 50 mg/kg (statistically significant at 50 mg/kg) were considered to be unrelated to treatment.

Treatment up to 150 mg/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
clinical biochemistry
gross pathology

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 150 mg/kg was derived.
The high dose of 150 mg/kg was considered to be the highest feasible dose in this type of study as in the acute oral toxicity study (Test Facility Study No. 515451), treatment-related hunched posture and mortality was observed for all animals within two hours after receiving 300 mg/kg.
Executive summary:

Title

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of CH02906 in rats by oral gavage.

Guidelines

The study was based on the following guidelines:

·     OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2016.

·     OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

·     OECD 421, Reproduction/Developmental Toxicity Screening Test, July 2016.

·     OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

·     EC No 440/2008 B.7: "Repeated Dose (28 days) Toxicity (oral)", May 2008.

·     OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.

·     OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

Rationale for dose levels

Based on the results of a dose range finder in which no dose limiting effects were noted at 150 mg/kg (Test Facility Study No. 515454) and acute oral toxicity data (oral LD50 between 50 and 300 mg/kg, Test Facility Study No. 515451), the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 15, 50 and 150 mg/kg.

Study outline

The test item, formulated in propylene glycol, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females.

Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were treated for 50-56 days (most females) or 62 days (one female), i.e. during 2 weeks prior to mating, during mating, duringpost-coitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were treated for 39-43 days.

Evaluated parameters

The following observations and examinations were evaluated: mortality/viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment and PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues.

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weight, anogenital distance, areola/nipple retention and macroscopy). Formulations were analyzed once during the study to assess accuracy and homogeneity.

Results

Accuracy and homogeneity of formulations were demonstrated by analyses.

Parental results:

No parental toxicity was observed up to the highest dose level tested (150 mg/kg).

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (150 mg/kg).

Developmental results:

No developmental toxicity was observed up to the highest dose level tested (150 mg/kg).

Conclusion

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 150 mg/kg was derived.

The high dose of 150 mg/kg was considered to be the highest feasible dose in this type of study as in the acute oral toxicity study, treatment-related hunched posture and mortality was observed for all animals within two hours after receiving 300 mg/kg.