Trans-2-[4'-[Difluor(3,4,5-trifluorphenoxy)methyl]-3',5'-difluor[1,1'-biphenyl]-4-yl]-5-propyl-tetrahydro-2H-pyran

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2017 - March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Remarks:

see section 7.8.2

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2017 - March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl: WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males were 10 weeks old and females were 13 weeks old
- Weight at study initiation: males weighed between 263 and 299 g and
females weighed between 213 and 239 g
- Fasting period before study: no
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, males were housed in their home cage with a maximum of 5 males/cage. Females were individually
housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam. The cages containing appropriate bedding (Lignocel S8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water: Municipal tap water was freely available to each animal via water bottles. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- Acclimation period: days prior to start of the pretest period (females) or 6 days before the commencement of dosing (males)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.9 to 21.5
- Humidity (%): 43 to 60
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 june 2017 To: 24 August 2017

Route of administration:

oral: gavage

Vehicle:

other: 0.25% (w/v) Aqueous hydroxypropyl methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. Test item dosing formulations (w/w) were homogenized with a blend er to visually acceptable
levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 6 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for concentration analysis during weeks 1, 3, 5 and the last week of treatment. The concentration results were obtained from the middle position of the preparations for Groups 1, 2, 3 and 4. The homogeneity results obtained from the top, middle and bottom for the Groups 2 and 4 preparations were averaged and utilized as the concentration results (week 1 of treatment). Analyses were performed by LC-UV using a validated analytical method.
concentration analysis: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable
if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration.
homogeneity analysis: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable
if the coefficient of variation (CV) of concentrations was equal or below 10%.
stability analysis: Stability analyses performed previously 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
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 post coitum
- A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.
- After successful mating each pregnant female was caged: During the post-mating phase, females were individually housed in Macrolon plastic cages

Duration of treatment / exposure:

Males were treated for 29 days and 37 days (one male which was used for additional mating of one female), i.e. up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-64 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), 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 (suspected) total litter loss were treated for 38-61 days.

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: first day of treatment (prior to dosing) and weekly thereafter. In addition, an extra measurement was performed on Day 12 of the pre-mating period based on the severe body weight loss observed in two high dose females on Day 8 pre-mating. 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. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was quantitatively measured weekly and, in addition, on Day 12 of the pre-mating period to monitor more closely the reduced food intake observed in Group 4 females from Days 1-8 pre-mating. No food consumption was recorded for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OTHER: blood samples were collected (end of treatment) for thyroid hormone measurement

Estrous cyclicity (parental animals)
Estrous cycles were 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.

Postmortem examinations (parental animals)
SACRIFICE
- Male animals: All surviving animals following completion of the mating period (a minimum of 28 days of administration).
- Females which delivered: PND 14-17
- Females which failed to deliver: With evidence of mating post-coitum Day 27.
Without evidence of mating 26 days after the last day of the mating period.
- Dams with no surviving pups were euthanized within 24 hours after the last pup is found dead or missing.
Except for females with confirmed total litter loss, all animals surviving to scheduled necropsy were fasted (overnight with a maximum of approximately 24 hours) before their scheduled necropsy. Water was available. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.
GROSS NECROPSY
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

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

Fetal examinations:

The following parameters were examined in F1 offspring: general health/mortality daily), clinical observations (at least once daily), body weights (PND 1, 4, 7 and 13), sex (PND 1 and 4), AGD (PND 1; normalized to the cube root of body weight), areola/nipple retention (PND 13); blood on PND 4 and PND 13-15 for measurement of thyroid hormone
gross examination of dead pups for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

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 matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
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:

Mating (%): (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 mated) x 100

Gestation index (%): (Number of females with living pups on Day 1 / Number of pregnant females) x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices
Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100

Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100

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

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations. Incidental findings that were noted 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.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female at 1000 mg/kg bw/day was sacrificed in extremis on Day 8 of the pre-mating period, based on severe body weight loss (-18%). Prior to early sacrifice, a hunched posture, piloerection and a lean appearance were observed for this female. At necropsy, no abnormalities were noted and at microscopic examination no cause of death could be determined.
No other mortality occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In females, slight to moderate body weight loss (average values of -1 to -5%) was observed at dose levels from 100 mg/kg bw/day onwards, showing a slight dose response, on pre-mating Days 8 and 12. This was generally followed by partial recovery on the first day of the mating period (i.e. treatment Day 15). From the start of the post-coitum period onwards until the end of the lactation period, absolute body weights were slightly lower at 300 and 1000 mg/kg bw/day when compared to controls, reaching statistical significance on post-coitum Days 14 and 17 for females at 1000 mg/kg bw/day ony (average values were 0.94x of controls). Body weight gain was unaffected by treatment during post-coitum and lactation for females at all dose levels.
Note: Body weight development during lactation at 1000 mg/kg bw/day could not fully be evaluated, as this phase included only one female. In males at 100 mg/kg bw/day, body weight gain was statistically significantly reduced on Days 8 and 15 of mating. On these days, body weight gain was also slightly lower for males at 300 and 1000 mg/kg bw/day, when compared to controls, but without reaching statistical significance. In the absence of a dose-related trend, no toxicological relevance was attached to this finding.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In females at 1000 mg/kg bw/day, food consumption before and after correction for body weight was slightly reduced (not statistically significant) on Days 1-12 of pre-mating, when compared to controls. During the post-coitum period, normal values were measured again, but during the lactation period, a trend towards slightly lower food intake (both absolute and relative to body weight) at 300 and 1000 mg/kg bw/day was seen, reaching statistical significance for females at 300 mg/kg bw/day on lactation Days 1-4 only.
Note: Food consumption during lactation at 1000 mg/kg bw/day could not fully be evaluated, as this phase included only one female. Food consumption before and after correction for body weight was similar to the control level in males up to and including 1000 mg/kg bw/day over the treatment period.

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Decreased levels of total T4 were observed in F0-males at all dose levels and F0-females at 1000 mg/kg bw/day.
Absolute mean values and relative changes to the concurrent control group are presented in the table in "any other information on results including tables."
In F0-males, mean values for total T4 at all dose levels were around the 5th percentile of the historical control range. At the individual level, T4 values in 6, 6 and 5 males at 100, 300 and 1000 mg/kg bw/day, respectively, were below the historical control range. Also in F0-females at 1000 mg/kg bw/day a significant decrease in total T4 was noted with a mean value below the 5th percentile of the historical range. It should be noted that at this highest dose tested a limited number of serum samples was available as five females had total litter loss. At the individual level, T4 values in 2 out of the 5 high dose females were clearly below the historical control range and one was at the lower end. In addition, one high dose female had a T4 value below the detection limit. Together with the observed lower mean values of total T4 in the 100 mg/kg bw/day (males), 300 mg/kg bw/day (males) and 1000 mg/kg bw/day (both sexes) groups also a trend towards lower mean values of TSH was noted compared to the concurrent control group. However, at the individual level there was no clear correlation between the direction of T4 and TSH levels, and the variation within each group was very high.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related alterations in organ weights.
The statistical significant changes noted for ovaries and uterus weights at 1000 mg/kg bw/day were caused by the different physiological status (i.e. total litter loss) compared to controls (i.e. lactating). This also caused the slightly increased mean uterus weight at 300 mg/kg bw/day in which 3/10 females had a total litter loss.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations. All 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.
Reproductive performance:
There were 4/10 couples at 300 mg/kg bw/day and 8/10 couples at 1000 mg/kg bw/day that were either not pregnant or had (suspected) total litter loss. For all females with (suspected) total litter loss there were implantation site(s) present in the uterus. For these females there also were other indicators of previous pregnancy, including luminal cell/blood debris in the uterus-cervix and/or vagina, increased mucification of the vagina and lobuloalveolar development of the mammary gland. For two animals with suspected total litter loss the implantation site(s) were older and there was no debris, indicating any delivery would have taken place longer before necropsy than in the other females with total litter loss. For the non-pregnant animal there was also an older implantation site present in the uterus, indicating this animal had been pregnant before.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).
Developmental toxicity was observed at 300 and 1000 mg/kg bw/day. Gestation index, postimplantation survival index, litter size and live birth index were reduced at both dose levels, in a dose related manner.
Gestation index was reduced to 78% at 300 mg/kg and 13% at 1000 mg/kg. Two females at 300 mg/kg bw/day and six females at 1000 mg/kg bw/day had only dead pups at first litter check or (suspected) total litter loss. In addition, one female at 1000 mg/kg bw/day was noted with an older implantation site only. This resulted in 7/9 and 1/8 pregnant females with living pups on Day 1 at 300 and 1000 mg/kg bw/day, respectively.

Number of abortions:

no effects observed

Description (incidence and severity):

No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was reduced to 61% at 300 mg/kg bw/day and 31% at 1000 mg/kg bw/day. It should be remarked that litter size was recorded on PND 1 and, consequently, any pups cannibalized prior to first litter check were not included in the total number of offspring born.
For three control females and two females at 100 mg/kg bw/day, the number of pups were slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 14-16 days of lactation. No toxicological relevance was attached to this finding in the current study.

Total litter losses by resorption:

no effects observed

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

The live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was reduced to 91% and 43% at 300 and 1000 mg/kg bw/day, respectively.
On an individual litter level at 300 mg/kg bw/day, one litter included 2 living pups and 3 dead pups, one litter had 1 dead pup and 7 living pups, one litter included 1 dead pup only, and one female had most likely cannibalised its pups before first litter check. At 1000 mg/kg bw/day, three litters had only dead pups at first litter check (with 2, 2 and 6 dead pups, respectively), three females had (most likely) cannibalised its pups before first litter check, and one litter consisted of 2 dead pups and 9 living pups.
The live birth index was not considered to be affected by treatment at 100 mg/kg bw/day.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Duration of gestation was not considered to be affected by treatment up to and including 1000 mg/kg bw/day.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

Fertility index was not considered to be affected by treatment. A slightly reduced fertility index was noted at 300 mg/kg bw/day and 1000 mg/kg bw/day (i.e. 90 and 89% versus 100%). For one of the females at 1000 mg/kg bw/day for which pregnancy was not confirmed, an older implantation site was found in the uterus at microscopic examination, indicating this animal had been pregnant. However, all values were
within normal limits.

Other effects:

no effects observed

Description (incidence and severity):

No deficiencies in maternal care were observed.

Key result

Dose descriptor:

NOAEL

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

Remarks:

Note: In this study, a marked reduction of total T4 was observed at 100, 300 mg/kg (males) and 1000 mg/kg (both sexes) which was considered to be compoundrelated. However, possible adversity of this effect could not be assessed within this type of screening study and was therefore not taken into account when determining the parental NOAEL.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Note: As only one litter survived beyond PND 1, data on 1000 mg/kg bw/day were not taken into account for the evaluation of pup body weights.
At 300 mg/kg bw/day, mean pup body weights were lower than those of controls from PND 7 onwards (relative differences (average of male and female pups) on PND 13 was 82% of controls).
Body weights of the 100 mg/kg bw/day pups were not considered to be affected by treatment.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

The live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was reduced to 91% and 43% at 300 and 1000 mg/kg bw/day, respectively.
On an individual litter level at 300 mg/kg bw/day, one litter included 2 living pups and 3 dead pups, one litter had 1 dead pup and 7 living pups, one litter included 1 dead pup only, and one female had most likely cannibalised its pups before first litter check. At 1000 mg/kg bw/day, three litters had only dead pups at first litter check (with 2, 2 and 6 dead pups, respectively), three females had (most likely) cannibalised its pups before first litter check, and one litter consisted of 2 dead pups and 9 living pups.
The live birth index was not considered to be affected by treatment at 100 mg/kg bw/day.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Note: As only one litter survived beyond PND 1, data on 1000 mg/kg bw/day were not taken into account for the evaluation of pup sex ratio.
Sex ratio at PND 1 appeared unaffected by treatment up to and including 300 mg/kg bw/day.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Litter size (mean number of living pups at first litter check) was reduced at 300 and 1000 mg/kg bw/day (6.6 and 2.3, respectively, versus 10.1 in controls). 1/8 Dams at 300 mg/kg bw/day and 3/4 dams at 1000 mg/kg bw/day had only dead pups. Litter size was not considered affected by treatment at 100 mg/kg bw/day.

At 300 mg/kg bw/day, mean pup body weights were lower than those of controls from PND 7 onwards (relative differences (average of male and female pups) on PND 13 was 82% of controls).
Body weights of the 100 mg/kg bw/day pups were not considered to be affected by treatment.
Note: As only one litter survived beyond PND 1, data on 1000 mg/kg bw/day were not taken into account for the evaluation of pup body weights.

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

Note: As only one litter survived beyond PND 1, data on 1000 mg/kg bw/day were not taken into account for the evaluation of viability index and the lactation index.
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment up to and including 300 mg/kg bw/day.
Three pups at 100 mg/kg bw/day (in two in litters) and four pups at 300 mg/kg bw/day (in two in litters) were found missing on PND 2-4. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

The lactation index (number of live offspring on Day 13 compared to the number of live offspring on Day 4 after culling) was not considered to be affected by treatment up to and including 300 mg/kg bw/day. One pup of the control group was found dead on PND 7 and one pup at 300 mg/kg bw/day was sacrificed in extremis on PND 8, based on its lean appearance. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

External malformations:

no effects observed

Description (incidence and severity):

Note: As only one litter survived beyond PND 1, data on 1000 mg/kg bw/day were not taken into account for the evaluation of pup macroscopic findings.
No macroscopic findings were noted among pups that were considered to be related to treatment up to and including 300 mg/kg bw/day. The nature and incidence of incidental macroscopic findings remained within the range considered normal for pups of this age, and were therefore not considered to be related to treatment.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Note: As only one litter survived beyond PND 1, data on 1000 mg/kg bw/day were not taken into account for the evaluation of pup T4 levels, anogenital distance and areola/nipple retention.
Serum T4 levels were statistically significantly decreased in female PND 13-15 pups at 300 mg/kg bw/day. The mean value at 300 mg/kg bw/day was 75% of the concurrent control female pups.
T4 levels of the 100 mg/kg bw/day pups (males and females) and 300 mg/kg bw/day male pups were not considered to be affected by treatment.
Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment up to and including 300 mg/kg bw/day.
Treatment up to and including 300 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Key result

Dose descriptor:

NOAEL

Effect level:

100 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/pup body weight changes

changes in litter size and weights

other: decreased serum T4 levels in female pups

Key result

Abnormalities:

not examined

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

Dose formulation analysis

Accuracy: In the control group formulations, no test item was detected. A small response at the retention time of the test item was observed in the chromatograms of the control group formulation prepared use in Week 5. The maximum contribution to the low dose group samples was 0.55% only, and thereforefor considered negligible.

The concentrations analyzed in the formulations of the low dose group, mid dose group and high dose group were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%; actual range: 88-109%).

For the formulation of the mid dose group prepared for use during the last week that all four dose groups were treated, the mean accuracy was above the target concentration (i.e. 123% of target). This slightly higher accuracy for the mid dose group formulation in the last week only was considered not to have any impact on the study data.

Homogeneity: The formulations of th low dose group and the high dose group were homogeneous (i.e. coefficient of variation ≤ 10%; actual range: 3.1-3.9%).

Thyroid hormone analyses:

	males				females
Dose level	cont	100	300	1000	cont	100	300	1000
Total T4 (μg/dL)	4.40	2.95	2.91	3.07	3.69	3.86	3.42	1.98**
T4 level relative to concurrent control (%)	n.a.	67	66	70	n.a.	105	93	54
TSH (μLU/mL)	0.273	0.176	0.148	0.197	0.231	0.285	0.309	0.119
TSH level relative to concurrent control (%)	n.a.	64	54	72	n.a.	123	134	52

**: P<0.01

Historical control data for males:

Total T4 (μg/dL): mean = 4.65; P5-P95 = 2.990-6.520 (n=715; period 2015- 2017).

TSH (μLU/mL): mean = 0.154; P5-P95 = 0.0440-0.4270 (n=65; period 2015-2017).

Historical control data for females:

Total T4 (μg/dL): mean = 3.40; P5-P95 = 1.700-5.060 (n=45; period 2015-2017).

TSH (μLU/mL): mean = 0.263; P5-P95 = 0.0390-0.7980 (n=30; period 2015-2017).

Conclusions:

Based on the results of this reproduction/developmental toxicity screening test with the test item, NOAELs for parental and reproduction toxicity of 1000 mg/kg bw/day were established. It should be noted that the observed marked reduction in total T4 at 100 and 300 mg/kg bw/day in males and and at 1000 mg/kg bw/day in both sexes was considered compound-related, though not taken into account for determination of the parental NOAEL as possible adversity of this effect could not be assessed in this screening study. Based on developmental toxicity observed at 300 and 1000 mg/kg bw/day (gestation index, post-implantation survival index, litter size, pup weight, live birth index, plasma t4 levels in female pups), a NOAEL of 100 mg/kg bw/day was established for developmental toxicity.

Executive summary:

A reproduction/developmental toxicity screening study was performed according to OECD guideline 421 and in accordance with GLP principles. Rats (10 sex/dose) were given orally by gavage the test item for a minimum of 28 days at dose levels 0, 100, 300 or 1000 mg/kg bw/day. The following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight and food consumption, estrous cycle length and regularity, serum levels of thyroxine (T4) and thyroid stimulating hormone (TSH) in both F0-males and F0-females, gross necropsy findings, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development

(mortality, clinical signs, body weights, anogenital distance, areola/nipple retention, serum level of thyroid hormone T4 in PND 13-15 pups, and macroscopy).

The formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

Parental results

One female at 1000 mg/kg bw/day was sacrificed in extremis on Day 8 of the pre-mating period, based on severe body weight loss (-18%). Prior to early sacrifice, a hunched posture, piloerection and a lean appearance were observed for this female. At necropsy, no abnormalities were noted and at microscopic examination no cause of death could be determined. At this single occurrence and as no similar findings during clinical observation were noted for the remaining animals, this finding was considered a chance finding and not treatment related.

Slight to moderate body weight loss (average values of -1 to -5%) was observed in females at dose levels from 100 mg/kg bw/day onwards on pre-mating Days 8 and 12, with correlating slightly reduced food intake in females at 1000 mg/kg bw/day. At 100 mg/kg bw/day, this recovered during the remainder of the treatment period. At 300 and 1000 mg/kg bw/day, body weights remained slightly lower however body weight gain was similar to control levels. Taken the slight nature of these findings and the observed recovery during treatment, these findings were not considered adverse.

In males, body weights and food intake were unaffected by treatment up to and including 1000 mg/kg bw/day.

Decreased mean values of total T4 were observed in F0-males at all dose levels (relative differences from control: 67, 66 and 70% at 100, 300 and 1000 mg/kg bw/day, respectively).and F0-females at 1000 mg/kg bw/day (relative difference from control: 54%). Together with the observed lower mean values of total T4 also a trend towards lower mean values of TSH was noted compared to the concurrent control group. However, at the individual level there was no clear correlation between the direction of T4 and TSH levels, and within each group variation in TSH values was relatively high. It should be noted that the marked reduction of total T4 occurred in the absence of corroborating changes in either organ weight or morphology of the thyroid gland. Given the magnitude of the observed decrease in total T4, it was considered to represent a compound-related effect. However, possible adversity of this effect could not be assessed within this type of screening study and was therefore not taken into account when determining the parental NOAEL.

No treatment-related toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, macroscopic examination, organ weights, and microscopic examination).

Reproductive results

No reproduction toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Developmental results

Developmental toxicity was observed at 300 and 1000 mg/kg bw/day. Gestation index, postimplantation survival index, litter size and live birth index were reduced at both dose levels, in a dose related manner. Gestation index was reduced to 78% at 300 mg/kg and 13% at 1000 mg/kg bw/day. Two females at 300 mg/kg bw/day and six females at 1000 mg/kg bw/day had only dead pups at first litter check or (suspected) total litter loss. In addition, one female at 1000 mg/kg bw/day was noted with an older implantation site only. This resulted in 7/9 and 1/8 pregnant females with living pups on Day 1 at 300 and 1000 mg/kg bw/day, respectively. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was reduced to 61% at 300 mg/kg bw/day and 31% at 1000 mg/kg bw/day. Litter size (mean number of living pups at first litter check) was dose related reduced at 300 and 1000 mg/kg bw/day (6.6 and 2.3, respectively, versus 10.1 in controls). The live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was reduced to 91% and 43% at 300 and 1000 mg/kg bw/day, respectively. No treatment-related changes up to and including 1000 mg/kg bw/day were noted for duration of gestation, parturition, and maternal care. Up to and including 300 mg/kg bw/day, no treatment-related changes were noted for viability index, lactation index, pup clinical signs, sex ratio, anogenital distance, areola/nipple retention, and macroscopic examination. As at 1000 mg/kg only one litter survived beyond PND 1, these parameters were not evaluated at this high dose. No developmental toxicity was observed at 100 mg/kg bw/day.

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following no-observed-adverse-effect levels (NOAEL) of the test item were established:

Parental NOAEL: at least 1000 mg/kg bw/day.

Note: In this study, a marked reduction of total T4 was observed at 100, 300 mg/kg bw/day (males) and 1000 mg/kg bw/day (both sexes) which was considered to be compound-related. However, possible adversity of this effect could not be assessed within this type of screening study and was therefore not taken into account when determining the parental NOAEL.

Reproduction NOAEL: at least 1000 mg/kg bw/day.

Developmental NOAEL: 100 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(2R,5S)-2-{4'-[difluoro(3,4,5-trifluorophenoxy)methyl]-3',5'-difluoro-[1,1'-biphenyl]-4-yl}-5-propyloxane

EC Number:

615-063-5

Cas Number:

700863-48-5

Molecular formula:

C27H23F7O2

IUPAC Name:

(2R,5S)-2-{4'-[difluoro(3,4,5-trifluorophenoxy)methyl]-3',5'-difluoro-[1,1'-biphenyl]-4-yl}-5-propyloxane

Test animals

Species:

rat

Strain:

other: 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. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist. This type of study plan was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee of Charles River Den Bosch as required by the Dutch Act on Animal Experimentation (February 1997).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males were 10 weeks old and females were 13 weeks old
- Weight at study initiation: males weighed between 263 and 299 g and
females weighed between 213 and 239 g
- Fasting period before study: no
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, males were housed in their home cage with a maximum of 5 males/cage. Females were individually
housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam. The cages containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water: Municipal tap water was freely available to each animal via water bottles. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- Acclimation period: days prior to start of the pretest period (females) or 6 days before the commencement of dosing (males)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.9 to 21.5
- Humidity (%): 43 to 60
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 june 2017 To: 24 August 2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.25% (w/v) Aqueous hydroxypropyl methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. Test item dosing formulations (w/w) were homogenized with a blender to visually acceptable
levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 6 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 post coitum
- A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.
- After successful mating each pregnant female was caged: During the post-mating phase, females were individually housed in Macrolon plastic cages

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for concentration analysis during weeks 1, 3, 5 and the last week of treatment. The concentration results were obtained from the middle position of the preparations for Groups 1, 2, 3 and 4. The homogeneity results obtained from the top, middle and bottom for the Groups 2 and 4 preparations were averaged and utilized as the concentration results (week 1 of treatment). Analyses were performed by LC-UV using a validated analytical method.
concentration analysis: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable
if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration.
homogeneity analysis: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable
if the coefficient of variation (CV) of concentrations was equal or below 10%.
stability analysis: Stability analyses performed previously 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 treated for 29 days and 37 days (one male which was used for additional mating of one female), i.e. up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-64 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), 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 (suspected) total litter loss were treated for 38-61 days.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
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.
The dose levels were selected based on information provided by the Sponsor, and in an attempt to produce graded responses to the test item. A nearly identical substance was tolerated in Wistar rats without any histopathological findings up to and including 1000 mg/kg bw/day in an OECD 407 study.
The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level was expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: first day of treatment (prior to dosing) and weekly thereafter. In addition, an extra measurement was performed on Day 12 of the pre-mating period based on the severe body weight loss observed in two high dose females on Day 8 pre-mating. 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. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was quantitatively measured weekly and, in addition, on Day 12 of the pre-mating period to monitor more closely the reduced food intake observed in Group 4 females from Days 1-8 pre-mating. No food consumption was recorded for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OTHER: blood samples were collected (end of treatment) for thyroid hormone measurement

Oestrous cyclicity (parental animals):

Estrous cycles were 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.

Sperm parameters (parental animals):

Parameters examined in all P males:
weights of testis, prostate, glans penis, LABC, Cowper's gland, epididymis and seminal vesicles.
For the testes of all males of the low dose and the high dose group, and all males that failed to sire a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: general health/mortality daily), clinical observations (at least once daily), body weights (PND 1, 4, 7 and 13), sex (PND 1 and 4), AGD (PND 1; normalized to the cube root of body weight), areola/nipple retention (PND 13); blood on PND 4 and PND 13-15 for measurement of thyroid hormone

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals following completion of the mating period (a minimum of 28 days of administration).
- Females which delivered: PND 14-16
- Females which failed to deliver: With evidence of mating post-coitum Day 27.
Without evidence of mating 26 days after the last day of the mating period.
- Dams with no surviving pups were euthanized within 24 hours after the last pup is found dead or missing.

Except for females with confirmed total litter loss, all animals surviving to scheduled necropsy were fasted (overnight with a maximum of approximately 24 hours) before their scheduled necropsy. Water was available. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

GROSS NECROPSY
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed: Epididymis, Gland coagulation, Glans Cowper’s, Gland parathyroid, Gland penis, Gland prostate, Gland seminal vesicle, Gland thyroid, Lavator ani plus bulbocavernosus muscle complex, Ovaries, Testes, Uterus.
Organs were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals euthanized in extremis.
Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.
Histopathological investigation was performed according to the Guideline

Postmortem examinations (offspring):

SACRIFICE
- On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible.
All remaining pups were euthanized on PND 13-15. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
In addition, blood was collected from two pups per litter if possible and the thyroid from two pups per litter (one male and one female pup, if possible) was
preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.

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 matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
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:

Mating (%): (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 mated) x 100

Gestation index (%): (Number of females with living pups on Day 1 / Number of pregnant females) x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100

Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations. Incidental findings that were noted 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.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female at 1000 mg/kg bw/day was sacrificed in extremis on Day 8 of the pre-mating period, based on severe body weight loss (-18%). Prior to early sacrifice, a hunched posture, piloerection and a lean appearance were observed for this female. At necropsy, no abnormalities were noted and at microscopic examination no cause of death could be determined.
No other mortality occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In females, slight to moderate body weight loss (average values of -1 to -5%) was observed at dose levels from 100 mg/kg bw/day onwards, showing a slight dose response, on pre-mating Days 8 and 12. This was generally followed by partial recovery on the first day of the mating period (i.e. treatment Day 15). From the start of the post-coitum period onwards until the end of the lactation period, absolute body weights were slightly lower at 300 and 1000 mg/kg bw/day when compared to controls, reaching statistical significance on post-coitum Days 14 and 17 for females at 1000 mg/kg bw/day ony (average values were 0.94x of controls). Body weight gain was unaffected by treatment during post-coitum and lactation for females at all dose levels.
Note: Body weight development during lactation at 1000 mg/kg bw/day could not fully be evaluated, as this phase included only one female.
In males at 100 mg/kg bw/day, body weight gain was statistically significantly reduced on Days 8 and 15 of mating. On these days, body weight gain was also slightly lower for males at 300 and 1000 mg/kg bw/day, when compared to controls, but without reaching statistical significance. In the absence of a dose-related trend, no toxicological relevance was attached to this finding.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In females at 1000 mg/kg bw/day, food consumption before and after correction for body weight was slightly reduced (not statistically significant) on Days 1-12 of pre-mating, when compared to controls. During the post-coitum period, normal values were measured again, but during the lactation period, a trend towards slightly lower food intake (both absolute and relative to body weight) at 300 and 1000 mg/kg bw/day was seen, reaching statistical significance for females at 300 mg/kg bw/day on lactation Days 1-4 only.
Note: Food consumption during lactation at 1000 mg/kg bw/day could not fully be evaluated, as this phase included only one female.
Food consumption before and after correction for body weight was similar to the control level in males up to and including 1000 mg/kg bw/day over the treatment period.

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Decreased levels of total T4 were observed in F0-males at all dose levels and F0-females at 1000 mg/kg bw/day.
Absolute mean values and relative changes to the concurrent control group are presented in the table in "any other information on results including tables."
In F0-males, mean values for total T4 at all dose levels were around the 5th percentile of the historical control range. At the individual level, T4 values in 6, 6 and 5 males at 100, 300 and 1000 mg/kg bw/day, respectively, were below the historical control range. Also in F0-females at 1000 mg/kg bw/day a significant decrease in total T4 was noted with a mean value below the 5th percentile of the historical range. It should be noted that at this highest dose tested a limited number of serum samples was available as five females had total litter loss. At the individual level, T4 values in 2 out of the 5 high dose females were clearly below the historical control range and one was at the lower end. In addition, one high dose female had a T4 value below the detection limit. Together with the observed lower mean values of total T4 in the 100 mg/kg bw/day (males), 300 mg/kg bw/day (males) and 1000 mg/kg bw/day (both sexes) groups also a trend towards lower mean values of TSH was noted compared to the concurrent control group. However, at the individual level there was no clear correlation between the direction of T4 and TSH levels, and the variation within each group was very high.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations. All 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.
Reproductive performance:
There were 4/10 couples at 300 mg/kg bw/day and 8/10 couples at 1000 mg/kg bw/day that were either not pregnant or had (suspected) total litter loss. For all females with (suspected) total litter loss there were implantation site(s) present in the uterus. For these females there also were other indicators of previous pregnancy, including luminal cell/blood debris in the uterus-cervix and/or vagina, increased mucification of the vagina and lobuloalveolar development of the mammary gland. For two animals with suspected total litter loss the implantation site(s) were older and there was no debris, indicating any delivery would have taken place longer before necropsy than in the other females with total litter loss. For the non-pregnant animal there was also an older implantation site present in the uterus, indicating this animal had been pregnant before.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were not considered to have been affected by treatment. Most females had regular cycles of 4 days. Extended di-estrus occurred in two control females, two females at 100 mg/kg bw/day, one female at 300 mg/kg bw/day and one female at 1000 mg/kg bw/day, all with a regular cycle. Given their incidental nature, absence of a dose-related incidence and/or absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating index was not considered to be affected by treatment. For one female at 1000 mg/kg bw/day, mating was not confirmed during in-life, and at necropsy no corpora lutea and no implantation sites (after Salewski staining) were observed macroscopically. However, at microscopic examination, an older implantation site was found to be present in the uterus, indicating this animal had been pregnant.
Precoital time was not considered to be affected by treatment. Most females mated within four days. There were two control females, two low dose females and one mid dose female that mated within 12-14 days. As no dose response was noted, it was not considered treatment related.
Number of implantation sites was not considered to be affected by treatment. The slightly lower mean value at 1000 mg/kg bw/day (9.7 versus 11.3 in controls) was due to two females (one had 4 implanations and another 8). As these individual values were also seen in the control group, it was not considered toxicologically relevant.
Fertility index was not considered to be affected by treatment. A slightly reduced fertility index was noted at 300 mg/kg bw/day and 1000 mg/kg bw/day (i.e. 90 and 89% versus 100%). For one of the females at 1000 mg/kg bw/day for which pregnancy was not confirmed, an older implantation site was found in the uterus at microscopic examination, indicating this animal had been pregnant. However, all values were within normal limits.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

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

Remarks:

In this study, a marked reduction of total T4 was observed at 100, 300 mg/kg (males) and 1000 mg/kg (both sexes) which was considered to be compoundrelated. However, possible adversity of this effect could not be assessed within this type of screening study and was therefore not taken into account when determining the parental NOAEL.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Note: As only one litter survived beyond PND 1, data on 1000 mg/kg were not taken into account for the evaluation of pup clinical signs.
In the 300 mg/kg bw/day group one female pup was observed with severely retarded growth. Its body weight was 5.5 gram on PND 8 which was approximately 50% lower compared to its litter mates on PND 7. A relation to treatment could not be excluded. Due to the poor condition of this pup it was euthanized on PND 8. No clinical signs occurred among pups that were considered to be related to treatment at
100 mg/kg bw/day.
The nature and incidence of incidental clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Note: As only one litter survived beyond PND 1, data on 1000 mg/kg bw/day were not taken into account for the evaluation of viability index.
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment up to and including 300 mg/kg bw/day. Three pups at 100 mg/kg bw/day (in two litters) and four pups at 300 mg/kg bw/day (in two in litters) were found missing on PND 2-4. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Note: As only one litter survived beyond PND 1, data on 1000 mg/kg bw/day were not taken into account for the evaluation of pup body weights.
At 300 mg/kg bw/day, mean pup body weights were lower than those of controls from PND 7 onwards (relative differences (average of male and female pups) on PND 13 was 82% of controls).
Body weights of the 100 mg/kg bw/day pups were not considered to be affected by treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Note: As only one litter survived beyond PND 1, data on 1000 mg/kg bw/day were not taken into account for the evaluation of pup T4 levels.
Serum T4 levels were statistically significantly decreased in female PND 13-15 pups at 300 mg/kg bw/day. The mean value at 300 mg/kg bw/day was 75% of the concurrent control female pups.
T4 levels of the 100 mg/kg bw/day pups (males and females) and 300 mg/kg bw/day male pups were not considered to be affected by treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Note: As only one litter survived beyond PND 1, data on 1000 mg/kg bw/day were not taken into account for the evaluation of pup macroscopic findings.
No macroscopic findings were noted among pups that were considered to be related to treatment up to and including 300 mg/kg bw/day.
The nature and incidence of incidental macroscopic findings remained within the range considered normal for pups of this age, and were therefore not considered to be related to treatment.

Other effects:

no effects observed

Description (incidence and severity):

Note: As only one litter survived beyond PND 1, data on 1000 mg/kg bw/day were not taken into account for the evaluation
Sex ratio at PND 1 and anogenital distance (absolute and normalized for body weight) in male and female pups appeared unaffected by treatment up to and including 300 mg/kg bw/day. Treatment up to and including 300 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Gestation index was reduced to 78% at 300 mg/kg bw/day and 13% at 1000 mg/kg bw/day. It was 100% in the control and 100 mg/kg bw/day group. One female at 300 mg/kg bw/day and three females at 1000 mg/kg bw/day had only dead pups at first litter check. In addition, one female at 300 mg/kg bw/day and one female at 1000 mg/kg bw/day had no pups at first litter check (these were most likely cannibalized before first litter check), and for two females at 1000 mg/kg bw/day total litter loss was suspected. Finally, one female at 1000 mg/kg bw/day was noted with an older implantation site in the uterus only. This resulted in a total of 7/9 and 1/8 pregnant females with living pups on Day 1 at 300 and 1000 mg/kg bw/day, respectively. At 1000 mg/kg bw/day, two females were suspected to have delivered but cannibalized their pups before a first litter check could have been performed. This assumption is based on the following observations: both females had implantation sites in their uterus and showed other microscopic indicators of previous pregnancy (luminal cell/blood debris in the uterus-cervix and/or vagina, increased mucification of the vagina and lobuloalveolar development of the mammary gland). In addition, during the period of gestation (Day 0-20 post-coitum) the increase in body weight was in the same range as usually seen for females with a normal pregnancy.
Duration of gestation was not considered to be affected by treatment up to and including 1000 mg/kg bw/day.

No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was reduced to 61% at 300 mg/kg bw/day and 31% at 1000 mg/kg bw/day. It should be remarked that litter size was recorded on PND 1 and, consequently, any pups cannibalized prior to first litter check were not included in the total number of offspring born.
For three control females and two females at 100 mg/kg bw/day, the number of pups were slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 14-16 days of lactation. No toxicological relevance was attached to this finding in the current study.

Litter size (mean number of living pups at first litter check) was reduced at 300 and 1000 mg/kg bw/day (6.6 and 2.3, respectively, versus 10.1 in controls). 1/8 Dams at 300 mg/kg bw/day and 3/4 dams at 1000 mg/kg bw/day had only dead pups. Litter size was not considered affected by treatment at 100 mg/kg bw/day.

The live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was reduced to 91% and 43% at 300 and 1000 mg/kg bw/day, respectively. On an individual litter level at 300 mg/kg bw/day, one litter included 2 living pups and 3 dead pups, one litter had 1 dead pup and 7 living pups, one litter included 1 dead pup only, and one female had most likely cannibalised its pups before first litter check. At 1000 mg/kg bw/day, three litters had only dead pups at first litter check with 2, 2 and 6 dead pups, respectively, three females had (most likely) cannibalised its pups before first litter check and one litter consisted of 2 dead pups and 9 living pups.

The lactation index (number of live offspring on Day 13 compared to the number of live offspring on Day 4 after culling) was not considered to be affected by treatment up to and including 300 mg/kg bw/day.
One pup of the control group was found dead on PND 7 and one pup at 300 mg/kg bw/day was sacrificed in extremis on PND 8, based on its lean appearance. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental toxicity was observed at 300 and 1000 mg/kg. Gestation index, postimplantation survival index, litter size and live birth index were reduced at both dose levels, in a dose related manner.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: general developmental toxicity (liter size and live birth index)

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Dose formulation analysis

Accuracy: In the control group formulations, no test item was detected. A small response at the retention time of the test item was observed in the chromatograms of the control group formulation prepared for use in Week 5. The maximum contribution to the low dose group samples was 0.55% only, and therefore considered negligible.

The concentrations analyzed in the formulations of the low dose group, mid dose group and high dose group were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%; actual range: 88-109%).

For the formulation of the mid dose group prepared for use during the last week that all four dose groups were treated, the mean accuracy was above the target concentration (i.e. 123% of target). This

slightly higher accuracy for the mid dose group formulation in the last week only was considered not to have any impact on the study data.

Homogeneity: The formulations of th low dose group and the high dose group were homogeneous (i.e. coefficient of variation ≤ 10%; actual range: 3.1-3.9%).

Thyroid hormone analyses:

	males				females
Dose level	cont	100	300	1000	cont	100	300	1000
Total T4 (μg/dL)	4.40	2.95	2.91	3.07	3.69	3.86	3.42	1.98**
T4 level relative to concurrent control (%)	n.a.	67	66	70	n.a.	105	93	54
TSH (μLU/mL)	0.273	0.176	0.148	0.197	0.231	0.285	0.309	0.119
TSH level relative to concurrent control (%)	n.a.	64	54	72	n.a.	123	134	52

  **: P<0.01

Historical control data for males:

Total T4 (μg/dL): mean = 4.65; P5-P95 = 2.990-6.520 (n=715; period 2015- 2017).

TSH (μLU/mL): mean = 0.154; P5-P95 = 0.0440-0.4270 (n=65; period 2015-2017).

Historical control data for females:

Total T4 (μg/dL): mean = 3.40; P5-P95 = 1.700-5.060 (n=45; period 2015-2017).

TSH (μLU/mL): mean = 0.263; P5-P95 = 0.0390-0.7980 (n=30; period 2015-2017).

Applicant's summary and conclusion

Conclusions:

Based on the results of this reproduction/developmental toxicity screening test with the test item, NOAELs for parental and reproduction toxicity of 1000 mg/kg bw/day were established. It should be noted that the observed marked reduction in total T4 at 100 and 300 mg/kg bw/day in males and and at 1000 mg/kg bw/day in both sexes was considered compound-related, though not taken into account for determination of the parental NOAEL as possible adversity of this effect could not be assessed in this screening study.

Executive summary:

A reproduction/developmental toxicity screening study was performed according to OECD guideline 421 and in accordance with GLP principles. Rats (10 sex/dose) were given orally by gavage the test item for a minimum of 28 days at dose levels 0, 100, 300 or 1000 mg/kg bw/day.

The following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight and food consumption, estrous cycle length and regularity, serum levels of thyroxine (T4) and thyroid stimulating hormone (TSH) in both F0-males and F0-females, gross necropsy findings, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, anogenital distance, areola/nipple retention, serum level of thyroid hormone T4 in PND 13-15 pups, and macroscopy).

The formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

Parental results

One female at 1000 mg/kg bw/day was sacrificed in extremis on Day 8 of the pre-mating period, based on severe body weight loss (-18%). Prior to early sacrifice, a hunched posture, piloerection and a lean appearance were observed for this female. At necropsy, no abnormalities were noted and at microscopic examination no cause of death could be determined. At this single occurrence and as no similar findings during clinical observation were noted for the remaining animals, this finding was considered a chance finding and not treatment related.

Slight to moderate body weight loss (average values of -1 to -5%) was observed in females at dose levels from 100 mg/kg bw/day onwards on pre-mating Days 8 and 12, with correlating slightly reduced food intake in females at 1000 mg/kg bw/day. At 100 mg/kg bw/day, this recovered during the remainder of the treatment period. At 300 and 1000 mg/kg bw/day, body weights remained slightly lower however body weight gain was similar to control levels. Taken the slight nature of these findings and the observed recovery during treatment, these findings were not considered adverse.

In males, body weights and food intake were unaffected by treatment up to and including 1000 mg/kg bw/day.

Decreased mean values of total T4 were observed in F0-males at all dose levels (relative differences from control: 67, 66 and 70% at 100, 300 and 1000 mg/kg bw/day, respectively).and F0-females at 1000 mg/kg bw/day (relative difference from control: 54%). Together with the observed lower mean values of total T4 also a trend towards lower mean values of TSH was noted compared to the concurrent control group. However, at the individual level there was no clear correlation between the direction of T4 and TSH levels, and within each group variation in TSH values was relatively high. It should be noted that the marked reduction of total T4 occurred in the absence of corroborating changes in either organ weight or morphology of the thyroid gland. Given the magnitude of the observed decrease in total T4, it was considered to represent a compound-related effect. However, possible adversity of this effect could not be assessed within this type of screening study and was therefore not taken into account when determining the parental NOAEL.

No treatment-related toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, macroscopic examination, organ weights, and microscopic examination).

Reproductive results

No reproduction toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the reproductive parameters investigated

in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Developmental results

Developmental toxicity was observed at 300 and 1000 mg/kg bw/day. Gestation index, postimplantation survival index, litter size and live birth index were reduced at both dose levels, in a dose related manner.

Gestation index was reduced to 78% at 300 mg/kg and 13% at 1000 mg/kg bw/day. Two females at 300 mg/kg bw/day and six females at 1000 mg/kg bw/day had only dead pups at first litter check or

(suspected) total litter loss. In addition, one female at 1000 mg/kg bw/day was noted with an older implantation site only. This resulted in 7/9 and 1/8 pregnant females with living pups on Day 1 at 300 and 1000 mg/kg bw/day, respectively. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was reduced to 61% at 300 mg/kg bw/day and 31% at 1000 mg/kg bw/day. Litter size (mean number of living pups at first litter check) was dose-related reduced at 300 and 1000 mg/kg bw/day (6.6 and 2.3, respectively, versus 10.1 in controls). The live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was reduced to 91% and 43% at 300 and 1000 mg/kg bw/day, respectively.

No treatment-related changes up to and including 1000 mg/kg bw/day were noted for duration of gestation, parturition, and maternal care.

Up to and including 300 mg/kg bw/day, no treatment-related changes were noted for viability index, lactation index, pup clinical signs, sex ratio, anogenital distance, areola/nipple retention, and macroscopic examination. As at 1000 mg/kg only one litter survived beyond PND 1, these parameters were not evaluated at this high dose.

No developmental toxicity was observed at 100 mg/kg bw/day.

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following no-observed-adverse-effect levels (NOAEL) of the test item were established:

Parental NOAEL: at least 1000 mg/kg bw/day.

Note: In this study, a marked reduction of total T4 was observed at 100, 300 mg/kg bw/day (males) and 1000 mg/kg bw/day (both sexes) which was considered to be compound-related. However, possible adversity of this effect could not be assessed within this type of screening study and was therefore not taken into account when determining the parental NOAEL.

Reproduction NOAEL: at least 1000 mg/kg bw/day.

Developmental NOAEL: 100 mg/kg bw/day.