1-Ethoxy-2,3-difluor-4-[4-(trans-4-propylcyclohexyl)-1-cyclohexen-1-yl]-benzene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 Jul 2018 - 06 Nov 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 Jul 2018 - 06 Nov 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (July 2000)

Version / remarks:

July 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: other guidance as listed under Principles of method if other than guideline

Principles of method if other than guideline:

In addition, the procedures described in this report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 2016);
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 10-11 weeks (males) and 13-14 weeks (females)
- Weight at study initiation: 275 - 314 g (males) or 192 - 223 g (females)
- Fasting period before study: no
- Housing: Pretest (females only) and pre-mating period: group housed (up to 5 animals of the same sex and same dosing group together in polycarbonate cages Macrolon, MIV type, height 18 cm). Mating phase: males and females were cohabitated on a one-to-one basis in Macrolon plastic cages (MIII type, height 18 cm). Post-mating phase: males were housed in their home cage with a maximum of 5 males/cage. Females were individually housed. Lactation phase: pups were housed with the dam (to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes). 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.
- Animal enrichment: For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
The actual daily mean temperature during the study period was 20 to 22 °C with an actual daily mean relative humidity of 51 to 78 %.

IN-LIFE DATES: From: 04 Jul 2018 to 07 Sept 2018

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Vehicle:

corn oil

Details on oral exposure:

Method of formulation: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Initially and based on the stability data, the dosing formulations were prepared at room temperature and protected from light in daily portions.

Storage conditions of formulations: If not used on the day of preparation, the formulations were stored in the refrigerator up to a maximum of eight days during the first two weeks of dosing and up to a maximum of 4 days during the remaining dosing period. On the day of use, the dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. All formulations were used for dosing within 6 hours after preparation, if used on the day of preparation, or within 6 hours after taken out of the refrigerator. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.

Justification for use and choice of vehicle (if other than water): Vehicle was selected based on trial experiments performed at the testing facility and based on information provided by the sponsor.

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed using a validated analytical procedure. Duplicate sets of samples (approximately 500 mg) 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 results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10 %. Stability analyses performed previously in conjunction with the method development and validation study. The results 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.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination.
Females were exposed for 50-65 days, i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 41-51 days. Females were not dosed if they were littering at the moment of dosing.

Frequency of treatment:

Once daily, 7 d/w

Dose / conc.:

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

Remarks:

Low dose

Dose / conc.:

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

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: The dose levels were selected based on the results of a 10-day dose range finder with oral administration of the test item in rats, and to determine the peak effect of occurrence of clinical signs after dosing. No guidelines were applicable as this study was intended for dose level selection purposes only. Test system, procedures and techniques were identical to those used during the main study. Three females were dosed with 500 and 1000 mg/kg bw/day, by once daily oral gavage for 10 consecutive days. The dose levels were selected based on information provided by the sponsor (Acute oral toxicity study in rats: LD50 > 2000 mg/kg bw; no deaths and no signs of toxicity observed up to 2000 mg/kg bw). Mortality was checked twice daily throughout the study. Clinical observations were done at least daily from Days 1-10, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing. Body weights were recorded on day 1 prior to dosing and on days 5 and 10 after dosing. Food consumption was monitored over days 1-5 and 5-10. All animals were subjected to an external, thoracic and abdominal examination on Day 11 (scheduled necropsy). Animals were not deprived of food prior to necropsy. Terminal body weight, kidney and liver weight were determined at scheduled necropsy. No organs were fixed and histopathological examination was not performed. No mortality or clinical signs were observed during the dose range finder study. Based on the results of the dose range finder, selected dose levels for the main study were 100, 300 and 1000 mg/kg bw/d. Since no clinical signs were observed in the dose range finder, it was decided to perform the clinical observations and functional observations directly after dosing in the main study.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
Yes
- Twice, daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS:
Yes
- Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.
- During the dosing period, these observations were at least performed directly after dosing based on the (absence of) clinical signs observed in the dose range finder.
- The time of onset, grade and duration of any observed signs were recorded using grades from 1 to 4, where 1 is slight and 4 is very severe. For certain signs, 2 scores were used: absence (grade 0) or presence (grade 1).

BODY WEIGHT:
Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure 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.

FOOD CONSUMPTION:
Yes. Weekly, for males and females. Food consumption was not recorded during the mating period. 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.

FOOD EFFICIENCY:
No.

WATER CONSUMPTION:
Yes, on regular basis throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled necropsia.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted:
Males: Yes, overnight (with a maximum of 24 hours). Water was provided.
Females: no
- Number of animals: 5 animals/sex/group
- Parameters checked: according to test guidelines.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled necropsia.
- Animals fasted:
Males: Yes, overnight (with a maximum of 24 hours). Water was provided.
Females: no
- Number of animals: 5 animals/sex/group
- Parameters checked: according to the guidelines.

THYROID HORMONES
- Measurement of total T4 and TSH was conducted for F0 males and females, and PND 14-16 pups.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Yes
- Time schedule for examinations: During Week 4 of treatment (males) and during the last week of lactation (females, i.e. PND 9-12). After dosing, after completion of clinical observations.
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex, and fore- and hind-limb grip strength (recorded as the mean of three measurements per animal) and locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system).
Total movements and ambulations were reported.
Deviations: Yes
- Locomotor activity test was performed twice for females as one female escaped during the first measurement.

Sacrifice and pathology:

SACRIFICE
- Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
- F0 males were fasted overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. F0 females were not fasted.
Scheduled euthanasias as follows:
- Males (which sired and failed to sire): after completion of mating period, minimum 28 days of administration.
- Females which delivered: PND 14-16
- Females which failed to deliver: Post coitum day 24 (with evidence of mating), 24 days after the last day of mating period (without evidence of mating)

GROSS PATHOLOGY: Yes
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
- The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

ORGAN WEIGHTS
- On selected 5 animals/sex/group: according to guidelines.
- All remaining animals: epididymis, prostate, seminal vesicles including coagulation glands, testes, thyroid.

HISTOPATHOLOGY: Yes
On selected 5 animals/sex/group in control and high-dose groups and all animals that were euthanized in extremis: according to guidelines.
- On selected 5 males in all groups and all males that failed to sire: additional testes slides.
- On all animals: all gross lesions.
- All remaining animals, including males that failed to sire and females that failed to deliver pups: Cervix, epididymis, glands (coagulation, mammary, parathyroid, pituitary, prostate, seminal vesicle, and thyroid), gross lesions/masses, ovaries, testes, uterus, vagina.

Other examinations:

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage (performed daily until evidence of copulation was observed or 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, except for females that had to be euthanized in extremis or died spontaneously.

Statistics:

All statistical tests were conducted at the 5 % significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5 % levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: Low dose group vs. control, Mid dose group vs. control, High dose group vs. control.
Parametric: Datasets with at least 2 groups (the designated control group and 1 other group) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric: Datasets with at least 2 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
Incidence: an overall Fisher’s exact test was used to compare all groups at the 5 % significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Piloerection was observed in a single female at 300 mg/kg bw/day dose group and among a few females at 1000 mg/kg bw/day during late post coitum and/or early lactation phase. Based on the incidence and time of occurrence these clinical signs were considered to be related to their pregnancy and maternal care, rather than being treatment related. The persistence of the piloerection in one female for a prolonged period was likely corresponding to the high mortality in its litter. Other clinical signs observed are considered incidental findings, i.e. scabs (control and 100 mg/kg bw/day), alopecia, hunched posture and red tears (chromodacryorrhoea; 1000 mg/kg bw/day). These findings 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. At the incidence observed, these were not considered to be signs of toxicological relevance. Salivation seen after dosing among animals of the 300 and 1000 mg/kg bw/day dose group from week 2-3 of the treatment period was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.

Mortality:

no mortality observed

Description (incidence):

One female of the 1000 mg/kg bw/day group was euthanized on lactation day 3, as she had a total litter loss.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significant increases observed for body weight gain in the 300 and 1000 mg/kg bw/day dose group females at lactation day 13, when compared to controls, were considered to be the result of relatively low body weight gain in the control group (and also in the 100 mg/kg bw/day dose group) (Historical control data females period 2015-2018: Body weight gain (%) lactation Day 13: mean: 14, P5-P95: 5.7-21.3, n=789). Since the body weight gain in the 300 and 1000 mg/kg bw/day dose group was within the normal range the statistical significances were a fortuitous finding and not related to treatment. The body weight values of two females (one on lactation day 7 and one on lactation day 13) were considered unrealistic and are probably weighing errors, based on the absence of corroborating clinical signs of ill health that might have explained the sudden body weight loss. Moreover, the terminal body weight for one female was in line with was to be expected based on its body weights during the lactation phase.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, the increase in food consumption during post coitum days 4-11 and the higher food consumption after correction for body weight during post coitum days 4-7 were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment and not of toxicological relevance.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

For males, the following changes in haematology were found: Decreased white blood cell count at 100 and 300 mg/kg bw/day (16 %, 19 %, respectively), decreased number of lymphocytes at 300 mg/kg bw/day (32 %), decreased haematocrit values at 100 mg/kg bw/day (5 %). Relative changes in mean values relative to the control group are indicated between parentheses. In absence of a dose response relationship, the alterations in haematological parameters were considered unrelated to administration of the test item. For females, no changes in haematological parameters were noted.

A decrease in prothrombin time was noted in males of 9 % compared with control at 100 mg/kg bw/day, which was considered unrelated to administration of the test item due to the minimal magnitude of the change and in absence of a dose response. In females, a reduction (not statistically significant) in activated partial thromboplastin time (APTT) appeared of 14 % compared with the control at 1000 mg/kg bw/day, which was considered unrelated to treatment as statistical significance was not reached and values were within normal range (Historical control data females period 2015-2018: Activated partial thromboplastin time: mean: 17.6; P5- P95: 13.1-22.7; n=136).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In absence of a dose response relationship, the statistical significances apparent for calcium levels in males treated at 300 mg/kg bw/day and for alkaline phosphatase and potassium levels in females treated at 100 mg/kg bw/day, in comparison with controls, were considered to have occurred by chance and not related to treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals.
Motor activity was similar between treated and control groups. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
The mean motor activity in control-group females was relatively low in comparison with the normal range for females of this age and strain (Total movements: mean: 3372, P5- P95: 1529-5618, n=400. Ambulations: mean: 817, P5-P95: 317-1440, n=400) and a large variation in motor activity was observed between the females in the low dose group (100 mg/kg bw/day). Two low dose females showed relatively high motor activity, for total movements as well as the ambulations, during the 7th to 10th (out of twelve) 5-min interval in comparison with the other three low dose females, resulting in a high standard deviation to the mean total movements and ambulation values for the low dose females. The results of the motor activity in the mid and high dose females, i.e. 300 and 1000 mg/kg bw/day respectively, were in the range as expected from the historical control data. In the absence of a dose response relationship and since all group mean values were within the normal range, the motor activity in females was considered not affected by treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant higher liver weights were noted in the 100, 300 and 1000 mg/kg bw/day group females.
One 100 mg/kg bw/day treated female had an abnormal papillary process of the liver (dark red discoloration and hardened), which was grown together with the stomach. This correlated microscopically with marked necrosis, some hemorrhage and a rim of granulation tissue around this lobe and was considered as an incidental finding. Since this was the only female of this group with an apparent higher individual liver weight, the significant liver weight change of the 100 mg/kg bw/day group was considered to be directly related to this single animal and unrelated to treatment with the test item.
The higher liver weights observed in 300 and 1000 mg/kg bw/day females were regarded as test item-related.
Any other differences were considered not to be test item-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Watery fluid in the uterus, found in two females of dose group 300 mg/kg bw/day is related to a stage in the estrous cycle and is a normal finding.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the liver and thyroid gland of the 300 and/or 1000 mg/kg bw/day group females. In the liver, hepatocellular hypertrophy was present at minimal degree in the 1000 mg/kg bw/day treated females. In the thyroid gland, an increased incidence and/or severity (up to mild) of follicular cell hypertrophy was present in 300 and 1000 mg/kg bw/day treated females. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid hormone analyses: Mean serum T4 levels of F0 males and F0 females were decreased by treatment at 100, 300 and 1000 mg/kg bw/day to a same degree in each sex when compared with the respective controls. The mean T4 levels were approximately 65 % lower in treated males (decreases of 63 %, 64 % and 6 8%, respectively) and approximately 35 % lower in treated females (decreases of 35 %, 38 % and 36 %, respectively), achieving statistical significance at all dose levels in each sex in comparison with controls. In treated males, the group mean values for T4 were below the historical control range at all dose levels tested (mean: 4.41, P5- P95: 2.76-6.34, n=333). In control females, a relatively high mean value for T4 of 3.55 μg/dL was observed in comparison with the historical mean value of 2.61 μg/dL (mean: 2.61, P5- P95: 1.56-4.040, n=48). However, because of the consistency of the decreases in T4 levels and a similar variation between the individual values in each treated group, the changes in T4 levels were considered to be treatment related, despite the fact that the absolute T4 levels in treated females were within the historical control range. The differences between the T4 levels of control and treated females within the current study were comparable to the difference between the values of the mean and lower limit of the historical control range (i.e. approximately 40 % from the mean). Mean serum TSH levels were considered not to be affected by treatment and no indication of a feedback response to the decreased T4 levels was apparent. Several relatively high TSH values were observed, but were not accompanied by low T4 levels in these individual animals. Furthermore, the distribution of these high TSH values over the dose groups also did not indicate a relation to treatment, but did affect the group mean values and corresponding standard deviation for this parameter.

Details on results:

In this study, a marked reduction of total T4 was observed in parental male and female rats and in PND 14-16 male and female pups at all three dose level of 100, 300 and 1000 mg/kg bw/day. Therefore, a no observed effect level (NOEL) could not be established in this study and was considered to be below the lowest dose level of 100 mg/kg bw/day. Furthermore, the possible impact of the changes in T4 could not be assessed within this type of screening study and was therefore not taken into account when determining the parental and developmental NOAEL.

Key result

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Analysis of dose preparations:

In the control group formulation, no test substance was detected. The concentrations analysed in the formulations of groups exposed to 100, 300 and 1000 mg/kg bw/day were in agreement with target concentrations (i.e. mean accuracies between 85 % and 115 %). The formulations of the low and high dose group were homogeneous (i.e. coefficient of variation ≤ 10 %).

Conclusions:

In a repeated dose oral toxicity study combined with a reproduction/developmental toxicity screening study with rats (OECD 422), adverse histopatological effects were observed in the thyroid gland of females at 300 and 1000 mg/kg bw/day. Therefore parental NOAEL was established to be at least 100 mg/kg bw/day based on the effects in the thyroid gland.

Executive summary:

A repeated dose oral toxicity study combined with a reproduction/developmental toxicity screening study with rats was performed according to OECD 422/EC guidelines and GLP principles. The test item was administered by daily oral gavage to male and female rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 50-65 days). In parental female rats, dose-related changes were observed in the liver at 1000 mg/kg bw/day, comprising increased hepatic weights and correlating hepatocellular hypertrophy, and in the thyroid gland at 300 and 1000 mg/kg bw/day, i.e. follicular cell hypertrophy. Corresponding changes were not observed up to the highest dose level in male rats. Furthermore, serum total T4 levels were approximately 65 % lower in males and approximately 35 % lower in females at all three dose levels when compared to controls. The findings in the liver were observed in female rats at 1000 mg/kg bw/day and were considered non-adverse. A linear relationship between the effects in the thyroid gland and the decreased T4 levels was not apparent in females. Effects on the thyroid gland in males were absent. In addition, treatment-related changes in TSH level were not observed in both sexes, and thus it is unlikely that the changes in thyroid gland and T4 levels were the result of a normal physiological feedback-regulation in the thyroid hormone homeostasis. In the worst case, the effects on the thyroid should be considered representative of an adverse effect, because they seemed not to be the result of a normal physiological feedback-regulation in the thyroid hormone homeostasis. The impact of the changes in T4 levels could not be evaluated within this type of screening study, because it is unknown if the decrease in total T4 (comprising free and protein-bound T4) had any effect on the metabolic active, free T4 concentration, which is responsible for the feedback-regulated thyroid homeostasis. Based on the histopatological effects observed in the thyroid gland, a parental no observed adverse effect level (NOAEL) for the test item was established to be 100 mg/kg bw/day.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 Jul 2018 - 06 Nov 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (July 2000)

Version / remarks:

July 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: other guidance as listed under Principles of method if other than guideline

Principles of method if other than guideline:

In addition, the procedures described in this report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 2016);
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Purity/Composition correction factor: No correction factor required.
Test item handling: Use amber glassware or wrap container in aluminium foil.

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 10-11 weeks (males) and 13-14 weeks (females)
- Weight at study initiation: 275 - 314 g (males) or 192 - 223 g (females)
- Fasting period before study: no
- Housing: Pretest (females only) and pre-mating period: group housed (up to 5 animals of the same sex and same dosing group together in polycarbonate cages Macrolon, MIV type, height 18 cm). Mating phase: males and females were cohabitated on a one-to-one basis in Macrolon plastic cages (MIII type, height 18 cm). Post-mating phase: males were housed in their home cage with a maximum of 5 males/cage. Females were individually housed. Lactation phase: pups were housed with the dam (to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes). 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.
- Animal enrichment: For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
The actual daily mean temperature during the study period was 20 to 22 °C with an actual daily mean relative humidity of 51 to 78 %.

IN-LIFE DATES: From: 04 Jul 2018 to 07 Sept 2018

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Method of formulation: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Initially and based on the stability data, the dosing formulations were prepared at room temperature and protected from light in daily portions.

Storage conditions of formulations: If not used on the day of preparation, the formulations were stored in the refrigerator up to a maximum of eight days during the first two weeks of dosing and up to a maximum of 4 days during the remaining dosing period. On the day of use, the dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. All formulations were used for dosing within 6 hours after preparation, if used on the day of preparation, or within 6 hours after taken out of the refrigerator. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.

Justification for use and choice of vehicle (if other than water): Vehicle was selected based on trial experiments performed at the testing facility and based on information provided by the sponsor.

Dose volume: 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 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
Length of cohabitation: A maximum of 14 days was allowed for mating.
Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/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. For one couple, detection of mating was not confirmed in first instance. The actual mating date was determined based on a re-evaluation of the vaginal lavage for presence of sperm cells. Consequently, this couple was separated one day after the actual mating date. The actual mating date was designated Day 0 post-coitum. After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed using a validated analytical procedure. Duplicate sets of samples (approximately 500 mg) 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 results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10 %. Stability analyses performed previously in conjunction with the method development and validation study. The results 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.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination.
Females were exposed for 50-65 days, i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 41-51 days. Females were not dosed if they were littering at the moment of dosing.

Frequency of treatment:

Once daily for 7 d/w.

Details on study schedule:

Age at mating of the mated animals in the study: Approximately 13 weeks

Dose / conc.:

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

Remarks:

Low dose

Dose / conc.:

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

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: The dose levels were selected based on the results of a 10-day dose range finder with oral administration of the test item in rats, and to determine the peak effect of occurrence of clinical signs after dosing. No guidelines were applicable as this study was intended for dose level selection purposes only. Test system, procedures and techniques were identical to those used during the main study. Three females were dosed with 500 and 1000 mg/kg bw/day, by once daily oral gavage for 10 consecutive days. The dose levels were selected based on information provided by the sponsor (Acute oral toxicity study in rats: LD50 > 2000 mg/kg bw; no deaths and no signs of toxicity observed up to 2000 mg/kg bw). Mortality was checked twice daily throughout the study. Clinical observations were done at least daily from Days 1-10, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing. Body weights were recorded on day 1 prior to dosing and on days 5 and 10 after dosing. Food consumption was monitored over days 1-5 and 5-10. All animals were subjected to an external, thoracic and abdominal examination on Day 11 (scheduled necropsy). Animals were not deprived of food prior to necropsy. Terminal body weight, kidney and liver weight were determined at scheduled necropsy. No organs were fixed and histopathological examination was not performed. No mortality or clinical signs were observed during the dose range finder study. Based on the results of the dose range finder, selected dose levels for the main study were 100, 300 and 1000 mg/kg bw/d. Since no clinical signs were observed in the dose range finder, it was decided to perform the clinical observations and functional observations directly after dosing in the main study.

Positive control:

No.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Yes
- Twice, daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS:
Yes
- Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.
- During the dosing period, these observations were at least performed directly after dosing based on the (absence of) clinical signs observed in the dose range finder.
- The time of onset, grade and duration of any observed signs were recorded using grades from 1 to 4, where 1 is slight and 4 is very severe. For certain signs, 2 scores were used: absence (grade 0) or presence (grade 1).

BODY WEIGHT:
Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure 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.

FOOD CONSUMPTION:
Yes. Weekly, for males and females. Food consumption was not recorded during the mating period. 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.

FOOD EFFICIENCY:
No.

WATER CONSUMPTION:
Yes, on regular basis throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled necropsia.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted:
Males: Yes, overnight (with a maximum of 24 hours). Water was provided.
Females: no
- Number of animals: 5 animals/sex/group
- Parameters checked: according to test guidelines.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled necropsia.
- Animals fasted:
Males: Yes, overnight (with a maximum of 24 hours). Water was provided.
Females: no
- Number of animals: 5 animals/sex/group
- Parameters checked: according to the guidelines.

THYROID HORMONES
- Measurement of total T4 and TSH was conducted for F0 males and females, and PND 14-16 pups.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Yes
- Time schedule for examinations: During Week 4 of treatment (males) and during the last week of lactation (females, i.e. PND 9-12). After dosing, after completion of clinical observations.
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex, and fore- and hind-limb grip strength (recorded as the mean of three measurements per animal) and locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system).
Total movements and ambulations were reported.
Deviations: Yes
- Locomotor activity test was performed twice for females as one female escaped during the first measurement.

PARTURITION AND MATERNAL CARE
Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth. 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.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage (performed daily until evidence of copulation was observed or 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, except for females that had to be euthanized in extremis or died spontaneously.

Sperm parameters (parental animals):

Slides of the testes were prepared for histopathological staging of spermatogenesis (5 males of the control and high dose group).

Litter observations:

The following parameters were examined in F1 offspring:
- Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.
- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on post natal days 1, 4, 7 and 13.
- Sex: Sex was determined for all pups on post natal days 1 and 4.
- Anogenital distance was measured on post natal day 1.
- Areola/nipple retention were examined on post natal day 13.

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

SACRIFICE
- Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
- F0 males were fasted overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. F0 females were not fasted.
Scheduled euthanasias as follows:
- Males (which sired and failed to sire): after completion of mating period, minimum 28 days of administration.
- Females which delivered: PND 14-16
- Females which failed to deliver: Post coitum day 24 (with evidence of mating), 24 days after the last day of mating period (without evidence of mating)

GROSS PATHOLOGY: Yes
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
- The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

ORGAN WEIGHTS
- On selected 5 animals/sex/group: according to guidelines.
- All remaining animals: epididymis, prostate, seminal vesicles including coagulation glands, testes, thyroid.

HISTOPATHOLOGY: Yes
On selected 5 animals/sex/group in control and high-dose groups and all animals that were euthanized in extremis: according to guidelines.
- On selected 5 males in all groups and all males that failed to sire: additional testes slides.
- On all animals: all gross lesions.
- All remaining animals, including males that failed to sire and females that failed to deliver pups: Cervix, epididymis, glands (coagulation, mammary, parathyroid, pituitary, prostate, seminal vesicle, and thyroid), gross lesions/masses, ovaries, testes, uterus, vagina.

Postmortem examinations (offspring):

SACRIFICE
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 14-16, except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20 %). The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded.
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.
For sacrificed pups particular attention was paid to the external reproductive genitals to examine signs of altered development.
Any abnormal pup, organ or tissue was preserved in 10 % buffered formalin for possible further examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
No.

Statistics:

All statistical tests were conducted at the 5 % significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5 % levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: Low dose group vs. control, Mid dose group vs. control, High dose group vs. control.
Parametric: Datasets with at least 2 groups (the designated control group and 1 other group) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric: Datasets with at least 2 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
Incidence: an overall Fisher’s exact test was used to compare all groups at the 5 % significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

For each group, the following calculations were performed:
- Mating index: (Number of females mated/Number of females paired) x 100
- Fertility index: (Number of pregnant females/Number of females paired) x 100
- Conception index: (Number of pregnant females/Number of females mated) x 100
- Gestation index: (Number of females bearing live pups/Number of pregnant females) x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

- Percentage live males at First Litter Check: (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check: (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Percentage of postnatal loss Days 0-4 of lactation: (Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check) x 100
- Viability index: (Number of live pups on Day 4 of lactation / Number of pups born alive) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Piloerection was observed in a single female at 300 mg/kg bw/day dose group and among a few females at 1000 mg/kg bw/day during late post coitum and/or early lactation phase. Based on the incidence and time of occurrence these clinical signs were considered to be related to their pregnancy and maternal care, rather than being treatment related. The persistence of the piloerection in one female for a prolonged period was likely corresponding to the high mortality in its litter. Other clinical signs observed are considered incidental findings, i.e. scabs (control and 100 mg/kg bw/day), alopecia, hunched posture and red tears (chromodacryorrhoea; 1000 mg/kg bw/day). These findings 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. At the incidence observed, these were not considered to be signs of toxicological relevance. Salivation seen after dosing among animals of the 300 and 1000 mg/kg bw/day dose group from week 2-3 of the treatment period was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.

Mortality:

no mortality observed

Description (incidence):

One female of the 1000 mg/kg bw/day group was euthanized on lactation day 3, as she had a total litter loss.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significant increases observed for body weight gain in the 300 and 1000 mg/kg bw/day dose group females at lactation day 13, when compared to controls, were considered to be the result of relatively low body weight gain in the control group (and also in the 100 mg/kg bw/day dose group) (Historical control data females period 2015-2018: Body weight gain (%) lactation Day 13: mean: 14, P5-P95: 5.7-21.3, n=789). Since the body weight gain in the 300 and 1000 mg/kg bw/day dose group was within the normal range the statistical significances were a fortuitous finding and not related to treatment. The body weight values of two females (one on lactation day 7 and one on lactation day 13) were considered unrealistic and are probably weighing errors, based on the absence of corroborating clinical signs of ill health that might have explained the sudden body weight loss. Moreover, the terminal body weight for one female was in line with was to be expected based on its body weights during the lactation phase.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, the increase in food consumption during post coitum days 4-11 and the higher food consumption after correction for body weight during post coitum days 4-7 were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment and not of toxicological relevance.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

For males, the following changes in haematology were found: Decreased white blood cell count at 100 and 300 mg/kg bw/day (16 %, 19 %, respectively), decreased number of lymphocytes at 300 mg/kg bw/day (32 %), decreased haematocrit values at 100 mg/kg bw/day (5 %). Relative changes in mean values relative to the control group are indicated between parentheses. In absence of a dose response relationship, the alterations in haematological parameters were considered unrelated to administration of the test item. For females, no changes in haematological parameters were noted.

A decrease in prothrombin time was noted in males of 9 % compared with control at 100 mg/kg bw/day, which was considered unrelated to administration of the test item due to the minimal magnitude of the change and in absence of a dose response. In females, a reduction (not statistically significant) in activated partial thromboplastin time (APTT) appeared of 14 % compared with the control at 1000 mg/kg bw/day, which was considered unrelated to treatment as statistical significance was not reached and values were within normal range (Historical control data females period 2015-2018: Activated partial thromboplastin time: mean: 17.6; P5- P95: 13.1-22.7; n=136).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In absence of a dose response relationship, the statistical significances apparent for calcium levels in males treated at 300 mg/kg bw/day and for alkaline phosphatase and potassium levels in females treated at 100 mg/kg bw/day, in comparison with controls, were considered to have occurred by chance and not related to treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals.
Motor activity was similar between treated and control groups. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
The mean motor activity in control-group females was relatively low in comparison with the normal range for females of this age and strain (Total movements: mean: 3372, P5- P95: 1529-5618, n=400. Ambulations: mean: 817, P5-P95: 317-1440, n=400) and a large variation in motor activity was observed between the females in the low dose group (100 mg/kg bw/day). Two low dose females showed relatively high motor activity, for total movements as well as the ambulations, during the 7th to 10th (out of twelve) 5-min interval in comparison with the other three low dose females, resulting in a high standard deviation to the mean total movements and ambulation values for the low dose females. The results of the motor activity in the mid and high dose females, i.e. 300 and 1000 mg/kg bw/day respectively, were in the range as expected from the historical control data. In the absence of a dose response relationship and since all group mean values were within the normal range, the motor activity in females was considered not affected by treatment.

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):

Test item-related microscopic findings were noted in the liver and thyroid gland of the 300 and/or 1000 mg/kg bw/day group females. In the liver, hepatocellular hypertrophy was present at minimal degree in the 1000 mg/kg bw/day treated females. In the thyroid gland, an increased incidence and/or severity (up to mild) of follicular cell hypertrophy was present in 300 and 1000 mg/kg bw/day treated females. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid hormone analyses: Mean serum T4 levels of F0 males and F0 females were decreased by treatment at 100, 300 and 1000 mg/kg bw/day to a same degree in each sex when compared with the respective controls. The mean T4 levels were approximately 65 % lower in treated males (decreases of 63 %, 64 % and 6 8%, respectively) and approximately 35 % lower in treated females (decreases of 35 %, 38 % and 36 %, respectively), achieving statistical significance at all dose levels in each sex in comparison with controls. In treated males, the group mean values for T4 were below the historical control range at all dose levels tested (mean: 4.41, P5- P95: 2.76-6.34, n=333). In control females, a relatively high mean value for T4 of 3.55 μg/dL was observed in comparison with the historical mean value of 2.61 μg/dL (mean: 2.61, P5- P95: 1.56-4.040, n=48). However, because of the consistency of the decreases in T4 levels and a similar variation between the individual values in each treated group, the changes in T4 levels were considered to be treatment related, despite the fact that the absolute T4 levels in treated females were within the historical control range. The differences between the T4 levels of control and treated females within the current study were comparable to the difference between the values of the mean and lower limit of the historical control range (i.e. approximately 40 % from the mean). Mean serum TSH levels were considered not to be affected by treatment and no indication of a feedback response to the decreased T4 levels was apparent. Several relatively high TSH values were observed, but were not accompanied by low T4 levels in these individual animals. Furthermore, the distribution of these high TSH values over the dose groups also did not indicate a relation to treatment, but did affect the group mean values and corresponding standard deviation for this parameter.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment. All females had regular cycles of 4 days.
No signs of difficult or prolonged parturition were noted among the pregnant females.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment. All females had regular cycles of 4 days.
Mating index was considered not to be affected by treatment. The mating indices were 90 % for the control and 100 % for the 100, 300 and 1000 mg/kg bw/day groups, respectively.
Precoital time was considered not to be affected by treatment. All females, with the exception of one female of 100 mg/kg bw/day, showed evidence of mating within 4 days.
Number of implantation sites was considered not to be affected by treatment.
Fertility index was considered not to be affected by treatment. The fertility indices were 100 %, 100 %, 80 % and 100 % for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
One female of the control and two females at 300 mg/kg bw/day were not pregnant. Since these cases of non-pregnancy showed no dose-related incidence across the dose groups, and given the absence of any reproductive/developmental toxicity, this was not considered to be related to treatment.

In this study, a marked reduction of total T4 was observed in parental male and female rats and in PND 14-16 male and female pups at all three dose level of 100, 300 and 1000 mg/kg bw/day. Therefore, a no observed effect level (NOEL) could not be established in this study and was considered to be below the lowest dose level of 100 mg/kg bw/day. Furthermore, the possible impact of the changes in T4 could not be assessed within this type of screening study and was therefore not taken into account when determining the parental and developmental NOAEL.

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment. For one pup (1000 mg/kg bw/day) who was missing on PND 3, less milk in the stomach was noted on PND 2.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Litter size was considered not to be affected by treatment. Live litter sizes were 10.9, 10.8, 11.4 and 10.0 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
At 1000 mg/kg bw/day, a high mortality was observed in two litters, comprising 15 pups that were found dead at first litter check. As a result of this high mortality a live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) of 87 % was calculated which was below the historical control range. The number of live offspring on Day 4 before culling compared with the number of offspring on Day 1 (viability index) and the number of live offspring on Day 13 after littering compared with the number of live offspring on Day 4 (lactation index) were considered not to be affected by treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were considered not to be affected by treatment.
From PND 7 onwards, body weights of pups appear (not statistically significant) slightly lower compared with their concurrent controls. As all values remained within normal ranges and no statistical significance was reached, this slight reduction in pup body weight was considered unrelated to treatment.

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:

effects observed, treatment-related

Description (incidence and severity):

At sacrifice on PND 14-16, the mean serum T4 levels in male and female pups were decreased at all dose levels in both sexes when compared to the respective controls. In males relative decreases of 47 %, 52 % and 56 % and in females of 40 %, 57 % and 52 % were observed at 100, 300 and 1000 mg/kg, respectively. All mean T4 levels achieved levels of statistical significance when compared with controls and were outside the historical control range (Historical control data Total T4 (μg/dL) period 2017-2018: Males: mean: 5.77, P5-P95: 4.25-7.77, n=276. Females: mean: 5.42, P5-P95: 3.96-6.96, n=275). Mean serum TSH levels were considered not to be affected by treatment. The changes in mean values for TSH in treated groups were within expectations from the normal variation observed in control PND 14-16 pups (Historical control data TSH (μU/mL) period 2017-2018: Males: mean: 0.140, P5-P95: 0.072-0.210, n=15. Females: mean: 0.126, P5-P95: 0.043-0.339, n=15).

Urinalysis findings:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 1000 mg/kg/day had no effect on areola/nipple retention. For none of the
examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.
A unilateral pelvic dilation in a single male pup from the 1000 mg/kg bw/day group was considered an incidental finding and not related to treatment.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Key result

Reproductive effects observed:

no

Analysis of dose preparations: In the control group formulation, no test substance was detected. The concentrations analysed in the formulations of groups exposed to 100, 300 and 1000 mg/kg bw/day were in agreement with target concentrations (i.e.mean accuracies between 85 % and 115 %). The formulations of the low and high dose group were homogeneous (i.e. coefficient of variation ≤ 10 %).

Conclusions:

Based on the results of this repeated dose oral toxicity study with the reproduction/developmental toxicity screening test, a parental no observed adverse effect level (NOAEL) for repeated dose toxicity for the test item was established to be 100 mg/kg bw/day, based on the effects observed in the thyroid gland. A reproduction and developmental NOAEL for parental animals was established at 1000 mg/kg bw/day, based on the absence of adverse effects up to and including the highest dose tested. The developmental NOAEL for the offspring was established at 300 mg/ kg bw/day, based on the mortality observed in the high dose group.

Executive summary:

A repeated dose oral toxicity study combined with a reproduction/developmental toxicity screening study with rats was performed according to OECD/EC guidelines and GLP principles. The test item was administered by daily oral gavage to male and female rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 50-65 days). In parental female rats, dose-related changes were observed in the liver, comprising increased hepatic weights and correlating hepatocellular hypertrophy, and in the thyroid gland, i.e. follicular cell hypertrophy, in parental female rats. Corresponding changes were not observed up to the highest dose level in male rats. Furthermore, serum total T4 levels were approximately 65 % lower in males and approximately 35 % lower in females at all three dose levels when compared to controls. The findings in the liver were observed in female rats at 1000 mg/kg bw/day and were considered non-adverse. A linear relationship between the effects in the thyroid gland and the decreased T4 levels was not apparent in females. Effects on the thyroid gland in males were absent. In addition, treatment-related changes in TSH level were not observed in both sexes, and thus it is unlikely that the changes in thyroid gland and T4 levels were the result of a normal physiological feedback-regulation in the thyroid hormone homeostasis. In the worst case, the effects on the thyroid should be considered representative of an adverse effect, because they seemed not to be the result of a normal physiological feedback-regulation in the thyroid hormone homeostasis. The impact of the changes in T4 levels could not be evaluated within this type of screening study, because it is unknown if the decrease in total T4 (comprising free and protein-bound T4) had any effect on the metabolic active, free T4 concentration, which is responsible for the feedback-regulated thyroid homeostasis.

A high mortality rate was observed in 2/10 litters at 1000 mg/kg bw/day, comprising 7/9 and 8/9 dead pups at first litter check. Based on these results, a relatively low live birth index of 87 % was calculated. Since such a high mortality rate was an unusual finding, the phenomenon was considered to be treatment related, and therefore indicative of developmental toxicity. Serum total T4 levels were decreased in male and female PND 14-16 pups at 100, 300 and 1000 mg/kg bw/day. The T4 levels were 47-56 % lower in male pups and 40-57 % lower in female pups in comparison with controls and all group mean values exceeded the lower limit of the historical control range. The impact of the changes in T4 levels the development of the pups could not be evaluated within this type of screening study. No treatment-related changes were noted in any of the other developmental parameters investigated in this study i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body eight, anogenital distance, areola/nipple retention, and macroscopic examination.

Based on the results of this repeated dose oral toxicity study with the reproduction/developmental toxicity screening test, a parental no observed adverse effect level (NOAEL) for repeated dose toxicity for the test item was established to be 100 mg/kg bw/day, based on the effects observed in the thyroid gland. A reproduction and developmental NOAEL for parental animals was established at 1000 mg/kg bw/day, based on the absence of adverse effects up to and including the highest dose tested. The developmental NOAEL for the offspring was established at 300 mg/ kg bw/day, based on the mortality observed in the high dose group.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Organisation of Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (July 2016)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (July 2000)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: other guidance as listed under Principles of method if other than guideline

Deviations:

no

Principles of method if other than guideline:

In addition, the procedures described in this report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 2016)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-ethoxy-2,3-difluoro-4-{4-[(1s,4r)-4-propylcyclohexyl]cyclohex-1-en-1-yl}benzene

EC Number:

603-006-7

Cas Number:

124728-62-7

Molecular formula:

C23H34F2O

IUPAC Name:

1-ethoxy-2,3-difluoro-4-{4-[(1s,4r)-4-propylcyclohexyl]cyclohex-1-en-1-yl}benzene

Test material form:

solid: crystalline

Specific details on test material used for the study:

Purity/Composition correction factor: No correction factor required.
Test item handling: Use amber glassware or wrap container in aluminium foil.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 10-11 weeks (males) and 13-14 weeks (females)
- Weight at study initiation: 275 - 314 g (males) or 192 - 223 g (females)
- Fasting period before study: no
- Housing: Pretest (females only) and pre-mating period: group housed (up to 5 animals of the same sex and same dosing group together in polycarbonate cages Macrolon, MIV type, height 18 cm). Mating phase: males and females were cohabitated on a one-to-one basis in Macrolon plastic cages (MIII type, height 18 cm). Post-mating phase: males were housed in their home cage with a maximum of 5 males/cage. Females were individually housed. Lactation phase: pups were housed with the dam (to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes). 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.
- Animal enrichment: For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
The actual daily mean temperature during the study period was 20 to 22 °C with an actual daily mean relative humidity of 51 to 78 %.

IN-LIFE DATES: From: 04 Jul 2018 to 07 Sept 2018

Administration / exposure

Route of administration:

oral: gavage

Details on exposure:

Method of formulation: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Initially and based on the stability data, the dosing formulations were prepared at room temperature and protected from light in daily portions.

Storage conditions of formulations: If not used on the day of preparation, the formulations were stored in the refrigerator up to a maximum of eight days during the first two weeks of dosing and up to a maximum of 4 days during the remaining dosing period. On the day of use, the dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. All formulations were used for dosing within 6 hours after preparation, if used on the day of preparation, or within 6 hours after taken out of the refrigerator. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.

Justification for use and choice of vehicle (if other than water): Vehicle was selected based in trial experiments performed at the testing facility and based on information provided by the sponsor.

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed using a validated analytical procedure. Duplicate sets of samples (approximately 500 mg) 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 results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10 %. Stability analyses performed previously in conjunction with the method development and validation study. The results 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.

Details on mating procedure:

M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates). Length of cohabitation: A maximum of 14 days was allowed for mating. Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or 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. For one couple, detection of mating was not confirmed in first instance. The actual mating date was determined based on a re-evaluation of the vaginal lavage for presence of sperm cells. Consequently, this couple was separated one day after the actual mating date. The actual mating date was designated Day 0 post-coitum. After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination.
Females were exposed for 50-65 days, i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 41-51 days. Females were not dosed if they were littering at the moment of dosing. Pups were not treated directly, but were potentially exposed to the test item in utero and through lactational transfer.

Frequency of treatment:

Once daily for 7 d/w.

Duration of test:

Males: 29 days
Females: 50-65 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Low dose

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Mid dose

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: The dose levels were selected based on the results of a 10-day dose range finder with oral administration of the test item in rats, and to determine the peak effect of occurrence of clinical signs after dosing. No guidelines were applicable as this study was intended for dose level selection purposes only. Test system, procedures and techniques were identical to those used during the main study. Three females were dosed with 500 and 1000 mg/kg bw/day, by once daily oral gavage for 10 consecutive days. The dose levels were selected based on information provided by the sponsor (Acute oral toxicity study in rats: LD50 > 2000 mg/kg bw; no deaths and no signs of toxicity observed up to 2000 mg/kg bw). Mortality was checked twice daily throughout the study. Clinical observations were done at least daily from Days 1-10, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing. Body weights were recorded on day 1 prior to dosing and on days 5 and 10 after dosing. Food consumption was monitored over days 1-5 and 5-10. All animals were subjected to an external, thoracic and abdominal examination on Day 11 (scheduled necropsy). Animals were not deprived of food prior to necropsy. Terminal body weight, kidney and liver weight were determined at scheduled necropsy. No organs were fixed and histopathological examination was not performed. No mortality or clinical signs were observed during the dose range finder study. Based on the results of the dose range finder, selected dose levels for the main study were 100, 300 and 1000 mg/kg bw/d. Since no clinical signs were observed in the dose range finder, it was decided to perform the clinical observations and functional observations directly after dosing in the main study.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS:
Yes
- Twice, daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS:
Yes
- Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.
- During the dosing period, these observations were at least performed directly after dosing based on the (absence of) clinical signs observed in the dose range finder.
- The time of onset, grade and duration of any observed signs were recorded using grades from 1 to 4, where 1 is slight and 4 is very severe. For certain signs, 2 scores were used: absence (grade 0) or presence (grade 1).

BODY WEIGHT:
Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure 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.

FOOD CONSUMPTION:
Yes. Weekly, for males and females. Food consumption was not recorded during the mating period. 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.

FOOD EFFICIENCY:
No.

WATER CONSUMPTION:
Yes, on regular basis throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled necropsia.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted:
Males: Yes, overnight (with a maximum of 24 hours). Water was provided.
Females: no
- Number of animals: 5 animals/sex/group
- Parameters checked: according to test guidelines.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled necropsia.
- Animals fasted:
Males: Yes, overnight (with a maximum of 24 hours). Water was provided.
Females: no
- Number of animals: 5 animals/sex/group
- Parameters checked: according to the guidelines.

THYROID HORMONES
- Measurement of total T4 and TSH was conducted for F0 males and females, and PND 14-16 pups.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Yes
- Time schedule for examinations: During Week 4 of treatment (males) and during the last week of lactation (females, i.e. PND 9-12). After dosing, after completion of clinical observations.
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex, and fore- and hind-limb grip strength (recorded as the mean of three measurements per animal) and locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system).
Total movements and ambulations were reported.
Deviations: Yes
- Locomotor activity test was performed twice for females as one female escaped during the first measurement.

ESTROUS CYCLES
- Evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed.
Vaginal lavage was 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, except for females that had to be euthanized in extremis or died spontaneously.

PARTURITION AND MATERNAL CARE
Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth. 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.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Blood sampling:

see above

Fetal examinations:

The following parameters were examined in F1 offspring:
- Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.
- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on post natal days 1, 4, 7 and 13.
- Sex: Sex was determined for all pups on post natal days 1 and 4.
- Anogenital distance was measured on post natal day 1.
- Areola/nipple retention were examined on post natal day 13.

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.

Statistics:

All statistical tests were conducted at the 5 % significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5 % levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: Low dose group vs. control, Mid dose group vs. control, High dose group vs. control.
Parametric: Datasets with at least 2 groups (the designated control group and 1 other group) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric: Datasets with at least 2 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
Incidence: an overall Fisher’s exact test was used to compare all groups at the 5 % significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Indices:

For each group, the following calculations were performed:
Reproductive indices:
- Mating index: (Number of females mated/Number of females paired) x 100
- Precoital time: Number of days between initiation of cohabitation and confirmation of mating
- Fertility index: (Number of pregnant females/Number of females paired) x 100
- Conception index: (Number of pregnant females/Number of females mated) x 100
- Gestation index: (Number of females bearing live pups/Number of pregnant females) x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
- Post implantation survival index: (Total number of offspring born/Total number of uterine implantation sites) x 100

Offspring indices
- Live birth index: (Number of live offspring on day 1 after littering/Total number of offspring born) x100
- Percentage live males at First Litter Check: (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check: (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Viability index: (Number of live offspring on day 4 before culling/Number live offspring on day 1 after littering) x 100
- Lactation index: (Number of live offspring on Day 13 after littering/Number live offspring on day 4 (after culling)) x 100

Historical control data:

Historical control data is indicated in results when necessary.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Piloerection was observed in a single female at 300 mg/kg bw/day dose group and among a few females at 1000 mg/kg bw/day during late post coitum and/or early lactation phase. Based on the incidence and time of occurrence these clinical signs were considered to be related to their pregnancy and maternal care, rather than being treatment related. The persistence of the piloerection in one female for a prolonged period was likely corresponding to the high mortality in its litter. Other clinical signs observed are considered incidental findings, i.e. scabs (control and 100 mg/kg bw/day), alopecia, hunched posture and red tears (chromodacryorrhoea; 1000 mg/kg bw/day). These findings 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. At the incidence observed, these were not considered to be signs of toxicological relevance. Salivation seen after dosing among animals of the 300 and 1000 mg/kg bw/day dose group from week 2-3 of the treatment period was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.

Mortality:

no mortality observed

Description (incidence):

One female of the 1000 mg/kg bw/day group was euthanized on lactation day 3, as she had a total litter loss.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significant increases observed for body weight gain in the 300 and 1000 mg/kg bw/day dose group females at lactation day 13, when compared to controls, were considered to be the result of relatively low body weight gain in the control group (and also in the 100 mg/kg bw/day dose group) (Historical control data females period 2015-2018: Body weight gain (%) lactation Day 13: mean: 14, P5-P95: 5.7-21.3, n=789). Since the body weight gain in the 300 and 1000 mg/kg bw/day dose group was within the normal range the statistical significances were a fortuitous finding and not related to treatment. The body weight values of two females (one on lactation day 7 and one on lactation day 13) were considered unrealistic and are probably weighing errors, based on the absence of corroborating clinical signs of ill health that might have explained the sudden body weight loss. Moreover, the terminal body weight for one female was in line with was to be expected based on its body weights during the lactation phase.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, the increase in food consumption during post coitum days 4-11 and the higher food consumption after correction for body weight during post coitum days 4-7 were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment and not of toxicological relevance.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

For females, no changes in haematological parameters were noted.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In absence of a dose response relationship, the statistical significances apparent for calcium levels in males treated at 300 mg/kg bw/day and for alkaline phosphatase and potassium levels in females treated at 100 mg/kg bw/day, in comparison with controls, were considered to have occurred by chance and not related to treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals.
Motor activity was similar between treated and control groups. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
The mean motor activity in control-group females was relatively low in comparison with the normal range for females of this age and strain (Total movements: mean: 3372, P5- P95: 1529-5618, n=400. Ambulations: mean: 817, P5-P95: 317-1440, n=400) and a large variation in motor activity was observed between the females in the low dose group (100 mg/kg bw/day). Two low dose females showed relatively high motor activity, for total movements as well as the ambulations, during the 7th to 10th (out of twelve) 5-min interval in comparison with the other three low dose females, resulting in a high standard deviation to the mean total movements and ambulation values for the low dose females. The results of the motor activity in the mid and high dose females, i.e. 300 and 1000 mg/kg bw/day respectively, were in the range as expected from the historical control data. In the absence of a dose response relationship and since all group mean values were within the normal range, the motor activity in females was considered not affected by treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant higher liver weights were noted in the 100, 300 and 1000 mg/kg bw/day group females.
One 100 mg/kg bw/day treated female had an abnormal papillary process of the liver (dark red discoloration and hardened), which was grown together with the stomach. This correlated microscopically with marked necrosis, some hemorrhage and a rim of granulation tissue around this lobe and was considered as an incidental finding. Since this was the only female of this group with an apparent higher individual liver weight, the significant liver weight change of the 100 mg/kg bw/day group was considered to be directly related to this single animal and unrelated to treatment with the test item.
The higher liver weights observed in 300 and 1000 mg/kg bw/day females were regarded as test item-related.
Any other differences were considered not to be test item-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Watery fluid in the uterus, found in two females of dose group 300 mg/kg bw/day is related to a stage in the estrous cycle and is a normal finding.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the liver and thyroid gland of the 300 and/or 1000 mg/kg bw/day group females. In the liver, hepatocellular hypertrophy was present at minimal degree in the 1000 mg/kg bw/day treated females. In the thyroid gland, an increased incidence and/or severity (up to mild) of follicular cell hypertrophy was present in 300 and 1000 mg/kg bw/day treated females. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid hormone analyses: Mean serum T4 levels of F0 males and F0 females were decreased by treatment at 100, 300 and 1000 mg/kg bw/day to a same degree in each sex when compared with the respective controls. The mean T4 levels were approximately 65 % lower in treated males (decreases of 63 %, 64 % and 6 8%, respectively) and approximately 35 % lower in treated females (decreases of 35 %, 38 % and 36 %, respectively), achieving statistical significance at all dose levels in each sex in comparison with controls. In treated males, the group mean values for T4 were below the historical control range at all dose levels tested (mean: 4.41, P5- P95: 2.76-6.34, n=333). In control females, a relatively high mean value for T4 of 3.55 μg/dL was observed in comparison with the historical mean value of 2.61 μg/dL (mean: 2.61, P5- P95: 1.56-4.040, n=48). However, because of the consistency of the decreases in T4 levels and a similar variation between the individual values in each treated group, the changes in T4 levels were considered to be treatment related, despite the fact that the absolute T4 levels in treated females were within the historical control range. The differences between the T4 levels of control and treated females within the current study were comparable to the difference between the values of the mean and lower limit of the historical control range (i.e. approximately 40 % from the mean). Mean serum TSH levels were considered not to be affected by treatment and no indication of a feedback response to the decreased T4 levels was apparent. Several relatively high TSH values were observed, but were not accompanied by low T4 levels in these individual animals. Furthermore, the distribution of these high TSH values over the dose groups also did not indicate a relation to treatment, but did affect the group mean values and corresponding standard deviation for this parameter.

Details on results:

In this study, a marked reduction of total T4 was observed in parental male and female rats and in PND 14-16 male and female pups at all three dose level of 100, 300 and 1000 mg/kg bw/day. Therefore, a no observed effect level (NOEL) could not be established in this study and was considered to be below the lowest dose level of 100 mg/kg bw/day. Furthermore, the possible impact of the changes in T4 could not be assessed within this type of screening study and was therefore not taken into account when determining the parental and developmental NOAEL.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The total number of offspring born compared with the total number of uterine implantations was considered not to be affected by treatment. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 91 %, 92 %, 90 %, 90 % for the control, 100, 300 and 1000 mg/kg groups, respectively.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Litter size was considered not to be affected by treatment.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Gestation index and duration of gestation were considered not to be affected by treatment. The gestation indices were 100 % for all groups.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

Fertility index was considered not to be affected by treatment. The fertility indices were 100 %, 100 %, 80 % and 100 % for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
One female of the control and two females at 300 mg/kg bw/day were not pregnant. Since these cases of non-pregnancy showed no dose-related incidence across the dose groups, and given the absence of any reproductive/developmental toxicity, this was not considered to be related to treatment.

Other effects:

no effects observed

Description (incidence and severity):

No signs of difficult or prolonged parturition were noted among the pregnant females.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: This effect is listed for the sake of completeness. The effect had no impact for reproductive/developmental performance of maternal animals.

2

Key result

Dose descriptor:

NOAEL

Remarks:

maternal developmental toxicity

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

not examined

Reduction in number of live offspring:

effects observed, non-treatment-related

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment. Male/female ratios were 46/54, 45/55, 55/45, 48/52 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights of pups were considered not to be affected by treatment. From PND 7 onwards, body weights of pups appear (not statistically significant) slightly lower compared with their concurrent controls. As all values remained within normal ranges and no statistical significance was reached, this slight reduction in pup body weight was considered unrelated to treatment.

Changes in postnatal survival:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, a high mortality was observed in two litters comprising 15 pups that were found dead at first litter check. As a result of this high mortality a live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) of 87 % was calculated which was below the historical control range (Historical control data females period 2015-2018: Live birth index (%): mean: 99, P5- P95: 96-100, n=98). The number of live offspring on Day 4 before culling compared with the number of offspring on Day 1 (viability index) and the number of live offspring on Day 13 after littering compared with the number of live offspring on Day 4 (lactation index) were considered not to be affected by treatment.

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid hormones: At sacrifice on PND 14-16, the mean serum T4 levels in male and female pups were decreased at all dose levels in both sexes when compared to the respective controls. In males relative decreases of 47 %, 52 % and 56 % and in females of 40 %, 57 % and 52 % were observed at 100, 300 and 1000 mg/kg bw/day, respectively. All mean T4 levels achieved levels of statistical significance when compared with controls and were outside the historical control range (Historical control data Total T4 (μg/dL) period 2017-2018: Males: mean: 5.77, P5-P95: 4.25-7.77, n=276. Females: mean: 5.42, P5-P95: 3.96-6.96, n=275). Mean serum TSH levels were considered not to be affected by treatment. The changes in mean values for TSH in treated groups were within expectations from the normal variation observed in control PND 14-16 pups (Historical control data TSH (μU/mL) period 2017-2018: Males: mean: 0.140, P5-P95: 0.072-0.210, n=15. Females: mean: 0.126, P5-P95: 0.043-0.339, n=15).

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no effects.
Remark: Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

 

Analysis of dose preparations: In the control group formulation, no test substance was detected. The concentrations analysed in the formulations of groups exposed to 100, 300 and 1000 mg/kg bw/day were in agreement with target concentrations (i.e.mean accuracies between 85 % and 115 %). The formulations of the low and high dose group were homogeneous (i.e. coefficient of variation ≤ 10 %).

Applicant's summary and conclusion

Conclusions:

Based on the results of this repeated dose oral toxicity study with the reproduction/developmental toxicity screening test, a parental no observed adverse effect level (NOAEL) for repeated dose toxicity for the test item was established to be 100 mg/kg bw/day, based on the effects observed in the thyroid gland. A reproduction and developmental NOAEL for parental animals was established at 1000 mg/kg bw/day, based on the absence of adverse effects up to and including the highest dose tested. The developmental NOAEL for the offspring was established at 300 mg/ kg bw/day, based on the mortality observed in the high dose group.

Executive summary:

A repeated dose oral toxicity study combined with a reproduction/developmental toxicity screening study with rats was performed according to OECD/EC guidelines and GLP principles. The test item was administered by daily oral gavage to male and female rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 50-65 days). In parental female rats, dose-related changes were observed in the liver, comprising increased hepatic weights and correlating hepatocellular hypertrophy, and in the thyroid gland, i.e. follicular cell hypertrophy, in parental female rats. Corresponding changes were not observed up to the highest dose level in male rats. Furthermore, serum total T4 levels were approximately 65 % lower in males and approximately 35 % lower in females at all three dose levels when compared to controls. The findings in the liver were observed in female rats at 1000 mg/kg bw/day and were considered non-adverse. A linear relationship between the effects in the thyroid gland and the decreased T4 levels was not apparent in females. Effects on the thyroid gland in males were absent. In addition, treatment-related changes in TSH level were not observed in both sexes, and thus it is unlikely that the changes in thyroid gland and T4 levels were the result of a normal physiological feedback-regulation in the thyroid hormone homeostasis. In the worst case, the effects on the thyroid should be considered representative of an adverse effect, because they seemed not to be the result of a normal physiological feedback-regulation in the thyroid hormone homeostasis. The impact of the changes in T4 levels could not be evaluated within this type of screening study, because it is unknown if the decrease in total T4 (comprising free and protein-bound T4) had any effect on the metabolic active, free T4 concentration, which is responsible for the feedback-regulated thyroid homeostasis.

A high mortality rate was observed in 2/10 litters at 1000 mg/kg bw/day, comprising 7/9 and 8/9 dead pups at first litter check. Based on these results, a relatively low live birth index of 87 % was calculated. Since such a high mortality rate was an unusual finding, the phenomenon was considered to be treatment related, and therefore indicative of developmental toxicity. Serum total T4 levels were decreased in male and female PND 14-16 pups at 100, 300 and 1000 mg/kg bw/day. The T4 levels were 47-56 % lower in male pups and 40-57 % lower in female pups in comparison with controls and all group mean values exceeded the lower limit of the historical control range. The impact of the changes in T4 levels the development of the pups could not be evaluated within this type of screening study. No treatment-related changes were noted in any of the other developmental parameters investigated in this study i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body eight, anogenital distance, areola/nipple retention, and macroscopic examination.

Based on the results of this repeated dose oral toxicity study with the reproduction/developmental toxicity screening test, a parental no observed adverse effect level (NOAEL) for repeated dose toxicity for the test item was established to be 100 mg/kg bw/day, based on the effects observed in the thyroid gland. A reproduction and developmental NOAEL for parental animals was established at 1000 mg/kg bw/day, based on the absence of adverse effects up to and including the highest dose tested. The developmental NOAEL for the offspring was established at 300 mg/ kg bw/day, based on the mortality observed in the high dose group.