4-[Difluor(3,4,5-trifluorphenoxy)methyl]-3,5-difluor-4'-propyl-1,1'-biphenyl

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 March 2017-10 August 2017

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:

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:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light

Species:

rat

Strain:

other: Crl:WI(Han)

Details on species / strain selection:

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: not applicable
- Age at study initiation: males: approximately 10-12 weeks, females: at pre-test approximately 9-10 weeks, at the start of treatment approximately 10-12 weeks.
- Weight at study initiation: males 247-296 g, females 196-240 g
- Fasting period before study: none
- Housing: general: sterilized sawdust was provided as bedding material and paper was provided as cage-enrichment/nesting material. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cages without cage-enrichment, bedding material, food and water.
Females:
Pre-test and pre-mating: 5 females/cage in Macrolon plastic cages
Mating: cohabitated on a 1:1 basis in Macrolon plastic cages
Post-mating: individually in Macrolon plastic cages
Lactation: in Macrolon plastic cages; pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
Males:
Pre-mating: 5 males/cage in Macrolon plastic cages
Mating: cohabitated on a 1:1 basis in Macrolon plastic cages
Post-mating: in their home cases, 5 animals/cage
- Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), except during motor activity measurements when animals were deprived of food and water for max. 2 hours.
- Water: free access to tap water, except during motor activity measurements when animals were deprived of food and water for max. 2 hours.
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 42-60
- Air changes (per hr): at least 10

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

Methocel® K4M; 0.25% (w/v) hydroxypropyl methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Preparation of 1000 mL vehicle
1. Approximately 1/3 of the required volume of Elix water was heated to at least 90°C.
2. 2.5 gram Methocel® K4M powder was added to the heated water with agitation.
3. The mixture was agitated until the particles were thoroughly wetted and evenly dispersed.
4. For complete solubilization, the remainder of the Elix water was added with agitation as cold water to lower the temperature of the dispersion. Once the dispersion reached the temperature at which the Methocel® K4M powder began to hydrate and viscosity increased.
5. Agitation was continued for at least 30 minutes.

Method of formulation:
Formulations (w/w) were prepared daily. The required amount of test item was weighed and the required amount of vehicle added. After homogenization with a blender, the container was wrapped in aluminium foil and the dose preparation was stirred on a magnetic stirrer at room temperature overnight (≥ 16 hours). No adjustment was made for specific gravity/density of the test item, vehicle, and/or formulation. No correction was made for the purity/composition of the test item.
The formulations were used within 4-6 hours after release by the formulation room.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on information provided by the sponsor and trial formulations performed at Charles River Den Bosch.
- Amount of vehicle (if gavage): 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1, avoiding sibling mating
- Length of cohabitation: until confirmed mating, for maximum 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post-coitum
- After successful mating each pregnant female was caged individually in Macrolon cages

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted during the treatment phase according to a validated method. Samples of dose preparations were taken after overnight stirring and released on the following morning. Samples of formulations were analyzed for homogeneity and accuracy of preparation. Stability in vehicle over 6 hours and 4 hours at room temperature under normal laboratory light conditions was also determined.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

Males: 29 days (2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy)
Females that delivered healthy offspring (controls only): 50-55 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy.
Females that failed to deliver healthy offspring (all females administered the test item) were treated for 39-44 days.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose, if possible

Dose / conc.:

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

Remarks:

Vehicle controls, Group 1

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on a dose range finding study in which male and female Wistar Han rats were treated by oral gavage for 28 days at dose levels of 0, 30, 60 and 100 mg/kg bw/day.
Treatment at 100 mg/kg bw/day caused severe toxicity. Next to mortality (one female on Day 8), clinical signs (piloerection, hunched posture), marked body weight loss together with severely reduced food consumption were noted during the first week of treatment, followed by partial recovery thereafter. No adverse effects were observed at 30 and 60 mg/kg bw/day during in-life. At end of treatment, changes in haematology and clinical biochemistry parameters were noted in all treated groups. These consisted of higher absolute WBC count together with a higher neutrophil count (males at 30, 60 and 100 mg/kg bw/day and females at 100 mg/kg bw/day), lower RBC count together with lower haemoglobin and haematocrit (females at 30, 60 and 100 mg/kg bw/day), a trend towards higher ASAT (males at 30, 60 and 100 mg/kg bw/day) and ALP (both sexes at 30, 60 and 100 mg/kg bw/day), and lower albumin (both sexes at 30, 60 and 100 mg/kg bw/day). At necropsy, all treated males had testes that were flaccid and except for two males at 30 mg/kg bw/day all testes were reduced in size. In addition, the epididymides of all males at 60 mg/kg bw/day were reduced in size. A trend towards higher liver weights (absolute and/or relative to body weight) was noted in males and females at 30, 60 and 100 mg/kg bw/day. Kidney weights were unaffected by treatment up to 100 mg/kg bw/day (both sexes).
- Rationale for animal assignment (if not random): based on the pre-test 40 females with at least two regular estrous cycles were selected at random and further used in the study.

Positive control:

Not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for mortality and viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals, at least 1 hour (± 30 min) after dosing (on the peak period of anticipated effects after treatment). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

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

FOOD CONSUMPTION: yes
- Time schedule for examinations: weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

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

OTHER:
HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: selected 5 animals/sex/group
- Parameters examined: WBC, differential leucocyte count, RBC, reticulocytes (as % RBC), RDW, haemoglobin, haematocrit, MCV, MCH, MCHC, platelets, PT, APTT.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy
- Animals fasted: Yes / No / Not specified
- How many animals: selected 5 animals/sex/group
- Parameters examined: ALAT, ASAT, ALP, total protein, albumin, total billirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: selected males during week 4 of treatment; selected females on PND 7-8 (females with live offspring, Group 1), PND 1 (females with total litter loss; Group 2) or once during Days 24-26 post-coitum (non-pregnant females and females with implantation sites only; Groups 3 and 4). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
- Dose groups that were examined: selected 5 animals/sex/group from all groups
- Battery of functions tested: sensory activity (heating, pupillary reflex, static righting reflex), grip strength (fore- and hind-limb), motor activity (total movements and ambulations)

Thyroid hormone analysis:
End of study from all animals at planned necropsy; this included females on Day 14-16 of lactation, all females that failed to deliver healthy pups and
all males after at least 4 weeks of treatment (including all males that failed to sire).
Note: Females of Groups 2-4 were sacrificed almost 2 weeks earlier than those of Group 1 due to the lack of offspring or early total litter loss of treated females
For males, 1 aliquot of 150 μL serum was used for measurement of thyroxine (T4), and the remaining volume of serum was stored for possible future measurement of thyroidstimulating hormone (TSH).
For females: The serum was stored for possible future measurement of thyroxine (T4) and/or thyroid-stimulating hormone (TSH).

Oestrous cyclicity (parental animals):

Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of
copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period.
During pretest, this was done for 48 females.
On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.

Sperm parameters (parental animals):

Parameters examined in the male parental generation:
testis weight, epididymis weight, seminal vesicles including coagulating glands weight, prostate weight.
The reproductive organs of the selected 5 males from all groups and all males that failed to sire to sire were examined by pathologist. Additional slides of the testes of the selected males of Groups 1 and 4 and all males that failed to sire were further examined to determine the staging of spermatogenesis.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible). Note: this was only performed for the control group pups, as no viable offspring was obtained in all test item-treated groups.
Blood samples were collected from two of the surplus pups for T4 determination. Selective elimination of pups, e.g. based upon body weight, or anogenital distance was not done. Whenever the number of male or female pups prevented 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:
mortality and viability (on PND 1 and daily thereafter); clinical signs (at least once daily); body weights (for live pups on PND 1, 4, 7 and 13); sex of pups (PND 1 and 4), stillbirths, live births, presence of gross anomalies, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups. Note: due to the lack of offspring or early total litter loss in all test item-treated groups the examinations could be performed only on the control group pups.

Thyroid hormone determination:
On PND 4 (at culling), blood was collected from two surplus pups per litter, if possible. If only 1 surplus pup per litter was available at culling, as much as possible blood was collected from this single pup.
Note: Only pups of the 0 mg/kg bw/day treatment group were available for blood collection at PND 4 (in the 5-50 mg/kg bw/day treatment groups there were no pups at PND 4 due to the lack of offspring or early total litter loss in these groups).
On PND 13-15 (scheduled necropsy), blood was collected from two pups per litter (controls only).


GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead. All pups of the females with total litter loss (except two litters) were collected and fixed in 10% buffered formalin for possible future examination. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: not performed

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: not performed

Postmortem examinations (parental animals):

SACRIFICE
Males: following completion of the mating period (a minimum of 28 days of dose administration).
Females that delivered: PND 14-16
Females that failed to deliver or with suspected total litter loss: Post-coitum Days 25-27 (females with evidence of mating) or approximately 24-26 days after the last day of the mating period (females without evidence of mating)
Females with total litter loss observed: within 24 hours from litter loss.

GROSS NECROPSY
After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following slides were examined by the pathologist:
• The preserved organs and tissues of the selected 5 animals/sex of the 0 mg/kg bw/day and the 50 mg/kg bw/day treatment groups.
• Additional slides of the testes of the selected 5 males of the 0 mg/kg bw/day and the 50 mg/kg bw/day treatment group and all males that failed to sire to examine staging of spermatogenesis.
• Thymus, thyroid gland, liver, spleen and reproductive organs from all selected 5 animals of the 5 and the 15 mg/kg bw/day treatment group, based on (possible) treatment-related changes in these organs in the 50 mg/kg bw/day treatment group.
• All gross lesions of all animals (all dose groups).
• The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups or had a total litter loss. In addition, histopathological examination of the mammary gland was conducted for the females that had total litter loss, except for the 15 mg/kg bw/day treatment group.
The following organ weights were recorded:
Selected 5 animals/sex/group: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles including coagulating glands, spleen, testes, thymus, thyroid including parathyroid if detectable, uterus including cervis.
All remaining animals: epididymides, prostate, seminal cesicles including coagulation glands, testes, tyroid including parathyroid if detectable.

Postmortem examinations (offspring):

SACRIFICE
- Culling: performed on day 4 postpartum, maximum of 8 pups/litter (4/sex/litter as nearly as possible).
The remaining pups were sacrificed on PND13-15 (controls only, as no live pups were born in test item-treated groups).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not performed.

Statistics:

The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Reproductive indices:

The following indices were calculated:
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 mated) x 100%
Gestation index (%) = (number of females with living pups on PND1/number of pregnant females) x 100%
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

The following indices were calculated:
Post-implantation survival index (%) = (total number of offspring born/total number of uterine implantation sites) x 100%
Live birth index (%) = (number of live offsprin on PND 1/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 PND4 before culling/number of live offspring on PND4) x 100%
Lactation index (%) = (number of live offspring on PND13/number of live offspring on PND 4 (after culling)) x 100%

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related clinical signs were observed in males up to 50 mg/kg bw/day.
In females, treatment-related clinical signs of toxicity were noted from 5 mg/kg bw/day onward, generally starting after about five weeks of treatment (i.e. towards the end of the post-coitum period). The main finding was piloerection which was noted in most females at 15 and 50 mg/kg bw/day and in one female at 5 mg/kg bw/day. Hunched posture was noted in one female at 15 mg/kg bw/day and three females at 50 mg/kg bw/day.
No additional clinical signs of toxicity were noted during the arena observations. Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

There were no premature decedents in both sexes. All test item treated females failed to deliver healthy pups and were sacrificed about 1 to 2 weeks before the control females.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males: body weight gain was slightly reduced, to about the same extent, at 15 and 50 mg/kg bw/day (statistically significant from Day 1 of the mating period). Mean body weights of these males did not differ statistically significantly from those of controls (relative difference at the end of the treatment period: about 5%).
Females: lower body weight gain occurred at 5 mg/kg bw/day throughout the pre-mating period (statistically significant), and at 15 and 50 mg/kg bw/day throughout the pre-mating and post-coitum periods (statistically significant on Day 1 of the mating period and from Day 14 or Day 17 of the post-coitum period). A finding of note was that all three non-pregnant females in the 15 mg/kg bw/day group and all six non-pregnant females of 50 mg/kg bw/day group lost 4-9% of their body weight from post-coitum Days 11-20. In addition, the two females in the 50 mg/kg bw/day group with implantation sites only lost 10% and 9% of their body weight from Days 14-20 post-coitum and Days 17-20 post-coitum, respectively. This body weight loss resulted in statistically significantly reduced body weights towards the end of the postcoitum period (relative differences from controls at post-coitum Day 20: 16% and 26% at 15 and 50 mg/kg bw/day, respectively).
The relatively low body weight gain in the post-coitum period noted for one female of Group 4 was attributed to the fact that she had 2 implantations only.
Note: Body weight development of lactating treated females could not be evaluated due to the complete lack of healthy offspring at 5, 15 and 50 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males: food consumption before or after correction for body weight was not affected by treatment up to 50 mg/kg bw/day.
Females: there was a trend towards lower food consumption before allowance for body weight from Day 7 of the post-coitum period at 15 and 50 mg/kg bw/day. The difference from controls was most marked and statistically significant at 50 mg/kg between Days 17-20. Food consumption after allowance for body weight was similar between treated and control females.
Note: Food consumption of lactating treated females could not be evaluated due to the lack of healthy offspring at 5, 15 and 50 mg/kg bw/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males:
• Lower haemoglobin, haematocrit and mean corpuscular volume (MCV) at 50 mg/kg bw/day (11%, 12% and 6%, respectively). The mean number of red blood cells at this dose level was 6% lower compared to controls (not statistically significant).
• Higher percentage of neutrophils from 5 mg/kg bw/day onward in a dose-related manner (50%, 79% and 98% at 5, 15 and 50 mg/kg bw/day, respectively; not statistically significant at 50 mg/kg bw/day). The corresponding absolute numbers of neutrophils were 15%, 54% and 62% higher compared to controls (not statistically significant).
• Higher percentage of monocytes from 5 mg/kg onward in a dose-related manner (70%, 123% and 185% at 5, 15 and 50 mg/kg bw/day, respectively; not statistically significant at 50 mg/kg bw/day). The corresponding absolute numbers of monocytes remained close to the concurrent control values.
• Lower total white blood cell (WBC) count (26%, 17% and 22%) and lower percentage of lymphocytes (8%, 14%, 17% ) at 5, 15 and 50 mg/kg bw/day, respectively. It should be noted that changes in treated males compared to controls did not reach statistical significance, were within normal limits or slightly below (relative lymphocyte count at 50 mg/kg bw/day), and control values were at the upper end of the historical range.

Females:
Note: Treated females had no healthy offspring and were sacrificed at post-coitum Day 25-27 (non-pregnant females and females with implantations only) or shortly after parturition (PND 1-2; females with total litter loss). In contrast, all control females were lactating and sacrificed at PND 14-16. Treated females selected for blood sampling had the following physiological status: five with total litter loss at 5 mg/kg bw/day; one non-pregnant and four with suspected total litter loss at 15 mg/kg bw/day; two non-pregnant, one with implantations only and two with suspected total litter loss at 50 mg/kg bw/day.
Several clinical pathology parameters are known to be influenced by physiological status (lactating versus nulliparous). This was taken into account when assessing the possible relation with treatment of differences in clinical pathology values between treated females and controls. Where useful, values in treated females were compared with historical control data from sub-chronic (90-day) oral toxicity studies with the strain of rats used in this study (rats were about 19 weeks old at the time of blood sampling). Relative changes in mean values compared to the concurrent control group are indicated between parentheses.
• Lower haemoglobin from 5 mg/kg bw/day onward (16%, 17% and 8% at 5, 15 and 50 mg/kg bw/day, respectively; latter difference not statistically significant).
• Lower mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) from 5 mg/kg bw/day onward (9% for MCH, 5-8% for MCHC).
• Lower mean values for the number of red blood cells and haematocrit at 5 and 15 mg/kg bw/day (about 10%, not statistically significant).
• Higher percentage of reticulocytes at 5 and 15 mg/kg bw/day (60% and 130%, respectively). The value in one 15 mg/kg bw/day female was about five-fold higher compared to control values.
• Higher red blood cell distribution width (RDW) at 5 mg/kg bw/day (135%).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Male rats:
• Higher alkaline phosphatase (ALP) at 15 and 50 mg/kg bw/day (74 and 86%, respectively).
• Lower albumin at 15 and 50 mg/kg bw/day (10 and 13%, respectively).
• Lower total protein at 50 mg/kg bw/day (5%).
• Lower bile acids at 15 and 50 mg/kg bw/day (45 and 44%, respectively).
• Lower creatinine from 5 mg/kg bw/day onward (10, 8 and 8% at 5, 15 and 50 mg/kg, respectively; not statistically significant at 50 mg/kg bw/day, but all mean values at the lower end or just below the historical range).

Females:
• Higher alkaline phosphatase (ALP) at 50 mg/kg bw/day (69%).
• Lower albumin at 15 and 50 mg/kg bw/day (7 and 14%, respectively).
• Lower total protein at 15 and 50 mg/kg bw/day (7% at both dose levels).
• Lower creatinine from 5 mg/kg bw/day onward (17% at 5 mg/kg bw/day, 20% at the higher dose levels).
The following statistically significant differences between treated females and concurrent controls were considered to be due to the difference in physiological status: lower values for alanine aminotransferase (ALAT) activity, cholesterol, bile acids, urea and inorganic phosphate, and higher values for sodium and chloride.
The other differences noted in treated females were considered unrelated to treatment due to somewhat high concurrent control values (aspartate aminotransferase (ASAT) activity) or because they occurred in the absence of a dose-related trend (lower ALP and higher fasting glucose at 5 mg/kg bw/day).
Thyroid hormone analyses (males only):
The serum T4 level of F0-males was statistically significantly decreased from 5 mg/kg bw/day onward (relative differences from controls: 24, 41 and 54% at 5, 15 and 50 mg/kg bw/day, respectively). Mean value for the 5 mg/kg bw/day group remained within the historical control range, but was clearly lower for the 15 and 50 mg/kg bw/day group. At the individual level, T4 values in a few 5 mg/kg bw/day males and in most males at the higher dose levels were below the historical control range.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly lower mean values for hind limb grip strength were noted at 15 and 50 mg/kg bw/day in both sexes (relative differences from controls: about 30-40%). Values in treated animals remained in the normal historical range for rats of this strain and age, except for those of two females in Group 3 and two females in Group 4 which were below this range.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
The lower motor activity noted in females treated at 5 mg/kg bw/day, particularly from test interval 4 onwards (i.e. >15 minutes), occurred in the absence of a dose-related trend and was therefore considered to be unrelated to treatment. One female at 15 mg/kg bw/day showed low motor activity throughout the 1-hour test period. This might be related to her health condition (she showed piloerection and had an abnormal pregnancy). It was noted that two other 15 mg/kg bw/day females and several 50 mg/kg bw/day females with piloerection and abnormal pregnancies showed normal motor activity. This female had normal outcomes for other measures in the neuromuscular domain (including gait, air righting reflex and grip strength). Therefore, her lower motor activity was considered not to represent a direct effect of the test item on this neuromuscular endpoint.

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 observed in the thymus, thyroid gland, liver, spleen, and male and female reproductive organs, as described below.
• Thymus: Increased apoptosis of lymphocytes was recorded in males starting at 15 mg/kg bw/day and in females starting at 5 mg/kg bw/day.
• Thyroid gland: An increased incidence and severity of hypertrophy of follicular cells was recorded in males starting at 15 mg/kg bw/day and in females at 50 mg/kg bw/day.
• Liver: An increased incidence and/or severity of centrilobular hepatocellular hypertrophy was recorded in males starting at 15 mg/kg bw/day.
• Spleen: An increased incidence and severity of extramedullary hematopoiesis was recorded in males and females at 50 mg/kg bw/day.
The high incidence and severity of extramedullary hematopoiesis in the spleen (and liver) of females at 5 and 15 mg/kg bw/day was considered to be mainly related to blood loss, due to abnormal pregnancies.
• Testes: Tubular degeneration/atrophy was recorded in a few males at 5 mg/kg bw/day and in all males at 15 and 50 mg/kg bw/day. In most males at 5 mg/kg bw/day and one male at 15 mg/kg bw/day sperm retention and/or tubular vacuolation, which are considered precursor findings of tubular degeneration/atrophy, were recorded. The PAS-stain showed no normal stages or all stages degenerative in all males at 15 and 50 mg/kg bw/day. In males of the control group and 5 mg/kg bw/day group all spermatogenic stages were present.
• Epididymides: Cell debris was recorded in a few males at 5 mg/kg bw/day and in all males at 15 and 50 mg/kg bw/day. Reduced sperm was recorded in all males at 15 and 50 mg/kg bw/day. These findings were regarded to be secondary to the degenerative changes in the testes.
• Uterus: Vascular necrosis at the implantation sites was recorded in six females at 5 mg/kg bw/day and one female at 15 mg/kg bw/day.
Hemorrhage in the uterine lumen and/or implantation sites was recorded in seven females at 5 mg/kg bw/day and three females at 15 mg/kg bw/day. The hemorrhage was considered to be related to the vascular necrosis and/or recent parturition.
There were no other test item-related histologic changes. 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

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

Treatment at 50 mg/kg bw/day resulted in abnormal estrous cycles in 7/10 females: one female (not pregnant) had an irregular cycle (persistent di-estrus); six females (not pregnant, implantations only or suspected total litter loss) had an acyclic cycle (at least 10 days without estrus). The remaining three females (all not pregnant) had regular cycles of 4 days.
At 15 mg/kg bw/day, one female (suspected total litter loss) had an irregular cycle (persistent di-estrus). An irregular cycle occasionally occurs at low incidence in untreated controls. However, taken in the context of the cycling abnormalities at 50 mg/kg bw/day, it cannot be excluded that the irregular cycle in this female was related to treatment.
Length and regularity of the estrous cycle were not affected at 5 mg/kg bw/day. All 5 mg/kg bw/day females had regular cycles of 4 days.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

The PAS-stain showed no normal stages or all stages degenerative in all males at 15 and 50 mg/kg bw/day. In males of the control group and 5 mg/kg bw/day group all spermatogenic stages were present.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

None of the treated animals had healthy offspring. All females of the control group delivered healthy pups.
At 5 mg/kg bw/day, all females were sacrificed because of (suspected) total litter loss. Vascular necrosis and/or hemorrhage at the implantation sites of the uterus as recorded in most of these females probably attributed to the lack of viability of these pups.
At 15 mg/kg bw/day total litter loss was suspected in 7/10 females. There was no histopathological correlate found for the total litter loss of 4 females. For three remaining females hemorrhage and/or vascular necrosis were present at the implantation sites, which probably attributed to the lack of viability of the pups of these females. 3/10 females were not pregnant. Tubular degeneration/atrophy in the testes was regarded as the cause of infertility for these non-pregnant couples.
At 50 mg/kg bw/day none of the couples delivered (healthy) offspring. Two couples had in life evidence for total litter loss, two couples showed implantation sites only and six couples were non-pregnant. No microscopic findings were seen in the female reproductive organs which could account for the lack of healthy offspring. Tubular degeneration/atrophy in the testes was regarded as the cause of infertility of the six non-pregnant couples.
Mating index was not affected by treatment. All females showed evidence of mating (mating index 100%).
Precoital time was considered not to be affected by treatment.
The numbers of implantation sites were decreased at 15 mg/kg bw/day (statistically significant) and 50 mg/kg bw/day. The mean numbers of implantation sites were 12.2, 11.5, 9.9 and 7.3 in the 0, 5, 15 and 50 mg/kg bw/day treatment groups, respectively (note: only four 50 mg/kg bw/day females had implantation sites).
Fertility index was reduced to 70% (7 pregnant females) at 15 mg/kg bw/day and 40% (4 pregnant females of which two females had implantation sites only) at 50 mg/kg bw/day.
Fertility index was unaffected by treatment at 5 mg/kg bw/day (100%).
Gestation index was 0% at 5, 15 and 50 mg/kg bw/day (compared to 100% in the concurrent control group). Duration of gestation was normal in all seven 5 mg/kg bw/day females that delivered (mean value of 22.0 days compared to 21.6 days in concurrent controls).

Key result

Dose descriptor:

LOAEL

Remarks:

reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (sperm measures)

reproductive performance

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

male reproductive system

Organ:

cauda epididymis

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

female reproductive system

Organ:

uterus

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 15 and 50 mg/kg bw/day no live pups were born.
In the 5 mg/kg bw/day group only 4/10 pregnant females had living pups at first litter check, but these pups had to be euthanized due to poor condition on the same day (PND 1).

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

At 15 and 50 mg/kg bw/day no live pups were born.
In the 5 mg/kg bw/day group only 4/10 pregnant females had living pups at first litter check, but these pups had to be euthanized due to poor condition on the same day (PND 1). Three females had only dead pups at first litter check. The remaining three females had no pups that could be evaluated: pups of one dam were cannibalized before first litter check, and for two females total litter loss was suspected based on available body weight data.

Body weight and weight changes:

not examined

Description (incidence and severity):

Due to the lack of viable offspring in all test item-treated groups effects on body weight could not be evaluated.

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:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: viability

Organ:

other: not applicable

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Reproductive effects observed:

yes

Lowest effective dose / conc.:

5 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

The concentrations analyzed in the formulations of the 5, 15 and 50 mg/kg bw/day treatment groups were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%; actual range: 97-102%, n = 6 for the 5 and 50 mg/kg bw/day treatment groups, n=2 for the 15 mg/kg bw/day treatment group) and prepared homogenously (i.e. coefficient of variation ≤ 10%; actual range: 1.2-6.1%), except for those of the 15 mg/kg bw/day treatment group prepared and analyzed on treatment day 2 (mean accuracy of 83%). This lower accuracy was attributed to inhomogeneity of the formulation (coefficient of variation was 30%). To facilitate the homogenization process, it was decided to keep all formulations for a longer period on the magnetic stirrer. Formulations prepared according to this revised protocol were used first from treatment Day 4 onward and both accuracy and homogeneity were confirmed (coefficient of variation 6.1%, mean accuracy 97% (n = 6)).

The formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 6 hours (mean accuracy of 86% and 101% for groups 2 and 4, respectively; n = 2 for both groups).

Conclusions:

In a GLP-compliant OECD guideline 422 study with rats treated by gavage, adverse test item-related effects were observed at the lowest dose level of 5 mg/kg bw/day. They were observed in reproductive organs, including testes (reduced weight, degeneration and atrophy together with tubular sperm retention), epididymides (luminal cell debris) and uterus (vascular necrosis of arteries at the implantation sites and hemorrhage in the uterine lumen and/or implantation sites). None of the test item-treated animals had healthy offspring. Only 4/10 females at the lowest dose level of 5 ppm had live pups; however, they were sacrificed in extremis at PND1. The fertility index was reduced to 70% at 15 mg/kg bw/day and 40% at 50 mg/kg bw/day. The numbers of implantation sites were decreased at 15 mg/kg bw/day and 50 mg/kg bw/day in a dose-related manner. Irregular estrous cycle was observed in females starting from 15 mg/kg bw/day, and no normal stages of spermatogenesis or all stages degenerative were observed in all males at 15 and 50 mg/kg bw/day. Based on these effects, the lowest dose level of 5 mg/kg bw/day was considered to be a LOAEL for reproductive and developmental toxicity.

Executive summary:

A classification and labelling for reproductive toxicity is considered based on the results of this study.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

5 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 March 2017-10 August 2017

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 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: OECD Guideline 421: Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3650: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3550: Reproduction/Developmental Toxicity Screening Test, July 2000

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light

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: not applicable
- Age at study initiation: males: approximately 10-12 weeks, females: at pre-test approximately 9-10 weeks, at the start of treatment approximately 10-12 weeks.
- Weight at study initiation: males 247-296 g, females 196-240 g
- Fasting period before study: none
- Housing: general: sterilized sawdust was provided as bedding material and paper was provided as cage-enrichment/nesting material. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cages without cage-enrichment, bedding material, food and water.
Females:
Pre-test and pre-mating: 5 females/cage in Macrolon plastic cages
Mating: cohabitated on a 1:1 basis in Macrolon plastic cages
Post-mating: individually in Macrolon plastic cages
Lactation: in Macrolon plastic cages; pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
Males:
Pre-mating: 5 males/cage in Macrolon plastic cages
Mating: cohabitated on a 1:1 basis in Macrolon plastic cages
Post-mating: in their home cases, 5 animals/cage
- Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), except during motor activity measurements when animals were deprived of food and water for max. 2 hours.
- Water: free access to tap water, except during motor activity measurements when animals were deprived of food and water for max. 2 hours.
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 42-60
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 March 2017 (allocation) To: 10 August 2017 (last data collected).

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.25% (w/v) hydroxypropyl methylcellulose

Details on exposure:

Preparation of 1000 mL vehicle
1. Approximately 1/3 of the required volume of Elix water was heated to at least 90°C.
2. 2.5 gram Methocel® K4M powder was added to the heated water with agitation.
3. The mixture was agitated until the particles were thoroughly wetted and evenly dispersed.
4. For complete solubilization, the remainder of the Elix water was added with agitation as cold water to lower the temperature of the dispersion. Once the dispersion reached the temperature at which the Methocel® K4M powder began to hydrate and viscosity increased.
5. Agitation was continued for at least 30 minutes.

Method of formulation:
Formulations (w/w) were prepared daily. The required amount of test item was weighed and the required amount of vehicle added. After homogenization with a blender, the container was wrapped in aluminium foil and the dose preparation was stirred on a magnetic stirrer at room temperature overnight (≥ 16 hours). No adjustment was made for specific gravity/density of the test item, vehicle, and/or formulation. No correction was made for the purity/composition of the test item.
The formulations were used within 4-6 hours after release by the formulation room.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on information provided by the sponsor and trial formulations performed at Charles River Den Bosch.
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted during the treatment phase according to a validated method. Samples of dose preparations were taken after overnight stirring and released on the following morning. Samples of formulations were analyzed for homogeneity and accuracy of preparation. Stability in vehicle over 6 hours and 4 hours at room temperature under normal laboratory light conditions was also determined.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Details on mating procedure:

- M/F ratio per cage: 1:1, avoiding sibling mating
- Length of cohabitation: until confirmed mating, for maximum 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post-coitum
- After successful mating each pregnant female was caged individually in Macrolon cages

Duration of treatment / exposure:

Males: 29 days (2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy)
Females that delivered healthy offspring (controls only): 50-55 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy.
Females that failed to deliver healthy offspring (all females administered the test item) were treated for 39-44 days.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose, if possible

Duration of test:

At least 29 days for males and 39-55 days for females

Dose / conc.:

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

Remarks:

Concurrent vehicle controls, Group 1

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on a dose range finding study in which male and female Wistar Han rats were treated by oral gavage for 28 days at dose levels of 0, 30, 60 and 100 mg/kg bw/day.
Treatment at 100 mg/kg bw/day caused severe toxicity. Next to mortality (one female on Day 8), clinical signs (piloerection, hunched posture), marked body weight loss together with severely reduced food consumption were noted during the first week of treatment, followed by partial recovery thereafter. No adverse effects were observed at 30 and 60 mg/kg bw/day during in-life. At end of treatment, changes in haematology and clinical biochemistry parameters were noted in all treated groups. These consisted of higher absolute WBC count together with a higher neutrophil count (males at 30, 60 and 100 mg/kg bw/day and females at 100 mg/kg bw/day), lower RBC count together with lower haemoglobin and haematocrit (females at 30, 60 and 100 mg/kg bw/day), a trend towards higher ASAT (males at 30, 60 and 100 mg/kg bw/day) and ALP (both sexes at 30, 60 and 100 mg/kg bw/day), and lower albumin (both sexes at 30, 60 and 100 mg/kg bw/day). At necropsy, all treated males had testes that were flaccid and except for two males at 30 mg/kg bw/day all testes were reduced in size. In addition, the epididymides of all males at 60 mg/kg bw/day were reduced in size. A trend towards higher liver weights (absolute and/or relative to body weight) was noted in males and females at 30, 60 and 100 mg/kg bw/day. Kidney weights were unaffected by treatment up to 100 mg/kg bw/day (both sexes).
- Rationale for animal assignment (if not random): based on the pre-test 40 females with at least two regular estrous cycles were selected at random and further used in the study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for mortality and viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals, at least 1 hour (± 30 min) after dosing (on the peak period of anticipated effects after treatment). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

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

FOOD CONSUMPTION: yes
- Time schedule for examinations: weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on PND 14-16 for females that delivered healthy offspring (control females only) or on post-coitum Days 25-27 (females with evidence of mating) or approximately 24-26 days after the last day of the mating period (females without evidence of mating).
Females with total litter loss observed: within 24 hours from litter loss.

- Organs examined:
The following organ weights were recorded:
Selected 5 animals/sex/group: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles including coagulating glands, spleen, testes, thymus, thyroid including parathyroid if detectable, uterus including cervis.
All remaining animals: epididymides, prostate, seminal cesicles including coagulation glands, testes, tyroid including parathyroid if detectable.

The following slides were examined by the pathologist:
• The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4
• Additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males that failed to sire to examine staging of spermatogenesis.
• Thymus, thyroid glands, liver, spleen and reproductive organs from all selected 5 animals of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
• All gross lesions of all animals (all dose groups).
• The reproductive organs6 of all males that failed to sire and all females that failed to deliver healthy pups or had a total litter loss. In addition, histopathological examination of the mammary gland was conducted for the females that had total litter loss, except for Group 3.

OTHER:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: selected 5 animals/sex/group
- Parameters examined: WBC, differential leucocyte count, RBC, reticulocytes (as % RBC), RDW, haemoglobion, gaematocrit, MCV, MCH, MCHC, platelets, PT, APTT.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy
- Animals fasted: Yes / No / Not specified
- How many animals: selected 5 animals/sex/group
- Parameters examined: ALAT, ASAT, ALP, total protein, albumin, total billirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: selected males during week 4 of treatment; selected females on PND 7-8 (females with live offspring, Group 1), PND 1 (females with total litter loss; Group 2) or once during Days 24-26 post-coitum (non-pregnant females and females with implantation sites only; Groups 3 and 4). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
- Dose groups that were examined: selected 5 animals/sex/group from all groups
- Battery of functions tested: sensory activity (heating, pupillary reflex, static righting reflex), grip strength (fore- and hind-limb), motor activity (total movements and ambulations)

Thyroid hormone analysis:
End of study from all animals at planned necropsy; this included females on Day 14-16 of lactation, all females that failed to deliver healthy pups and
all males after at least 4 weeks of treatment (including all males that failed to sire).
Note: Females of Groups 2-4 were sacrificed almost 2 weeks earlier than those of Group 1 due to the lack of offspring or early total litter loss of treated females
For males, 1 aliquot of 150 μL serum was used for measurement of thyroxine (T4), and the remaining volume of serum was stored for possible future measurement of thyroidstimulating hormone (TSH).
For females: The serum was stored for possible future measurement of thyroxine (T4) and/or thyroid-stimulating hormone (TSH).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: no, the females were allowed to litter naturally and sacrificed on PND 14-16 (for females with healthy offspring) or on post-coitum Days 25-27 (females with evidence of mating) or approximately 24-26 days after the last day of the mating period (females without evidence of mating). Females with total litter loss observed were sacrificed within 24 hours from litter loss.
Examinations included:
- Gravid uterus weight: no, females were allowed to litter normally.
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

No fetal examinations were performed, as females were allowed to litter naturally.
The following parameters were examined in F1 offspring (note: only control animals had healthy offspring, there was no viable offspring in all test item-treated groups).
Mortality and viability: PND1 and daily thereafter.
Clinical signs: at least once daily
Body weights: for live pups on PND 1, 4, 7, 13.
Sex: for all pups on PND 1 and 4
Anogenital distance: for all live pups on PND 1, normalized to the cube root of body weight. Note: only pups of Groups 1 and 2 were available for this examination; females of Groups 3 and 4 had no offspring.
Areola/nipple retention: all males, on PND 13. Note: only pups from the control group were available for the examination.
Blood sampling for thyroid hormone analysis (note: only pups from the control group were available for culling and blood sampling):
PND4: if possible, from 2 surplus pups per litter at culling. If only 1 surplus pup per litter was available at culling, as much as possible blood was collected from this single pup.
PND 13-15: from 2 pups per litter, if possible, 1 male and 1 female, at scheduled necropsy

Statistics:

The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Indices:

The following indices were calculated:
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 mated) x 100%
Gestation index (%) = (number of females with living pups on PND1/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%
Live birth index (%) = (number of live offsprin on PND 1/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 PND4 before culling/number of live offspring on PND4) x 100%
Lactation index (%) = (number of live offspring on PND13/number of live offspring on PND 4 (after culling)) x 100%

Historical control data:

Historical control data are available for the period of 2015 - June 2017.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In females, treatment-related clinical signs of toxicity were noted from 5 mg/kg bw/day onward, generally starting after about five weeks of treatment (i.e. towards the end of the post-coitum period). The main finding was piloerection which was noted in most females at 15 and 50 mg/kg bw/day and in one female at 5 mg/kg bw/day. Hunched posture was noted in one female at 15 mg/kg bw/day and three females at 50 mg/kg bw/day.
No additional clinical signs of toxicity were noted during the arena observations. Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

There were no premature deaths. All test item treated females failed to deliver healthy pups and were sacrificed about 1 to 2 weeks before the control females.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Females: lower body weight gain occurred at 5 mg/kg bw/day throughout the pre-mating period (statistically significant), and at 15 and 50 mg/kg bw/day throughout the pre-mating and post-coitum periods (statistically significant on Day 1 of the mating period and from Day 14 or Day 17 of the post-coitum period). A finding of note was that all three non-pregnant females in the 15 mg/kg bw/day group and all six non-pregnant females of 50 mg/kg bw/day group lost 4-9% of their body weight from post-coitum Days 11-20. In addition, the two females in the 50 mg/kg bw/day group with implantation sites only lost 10% and 9% of their body weight from Days 14-20 post-coitum and Days 17-20 post-coitum, respectively. This body weight loss resulted in statistically significantly reduced body weights towards the end of the postcoitum period (relative differences from controls at post-coitum Day 20: 16% and 26% at 15 and 50 mg/kg bw/day, respectively).
The relatively low body weight gain in the post-coitum period noted for one female of Group 4 was attributed to the fact that she had 2 implantations only.
Note: Body weight development of lactating treated females could not be evaluated due to the complete lack of healthy offspring at 5, 15 and 50 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Females: there was a trend towards lower food consumption before allowance for body weight from Day 7 of the post-coitum period at 15 and 50 mg/kg bw/day. The difference from controls was most marked and statistically significant at 50 mg/kg between Days 17-20. Food consumption after allowance for body weight was similar between treated and control females.
Note: Food consumption of lactating treated females could not be evaluated due to the lack of healthy offspring at 5, 15 and 50 mg/kg bw/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Females:
Note: Treated females had no healthy offspring and were sacrificed at post-coitum Day 25-27 (non-pregnant females and females with implantations only) or shortly after parturition (PND 1-2; females with total litter loss). In contrast, all control females were lactating and sacrificed at PND 14-16. Treated females selected for blood sampling had the following physiological status: five with total litter loss at 5 mg/kg bw/day; one non-pregnant and four with suspected total litter loss at 15 mg/kg bw/day; two non-pregnant, one with implantations only and two with suspected total litter loss at 50 mg/kg bw/day.
Several clinical pathology parameters are known to be influenced by physiological status (lactating versus nulliparous). This was taken into account when assessing the possible relation with treatment of differences in clinical pathology values between treated females and controls. Where useful, values in treated females were compared with historical control data from sub-chronic (90-day) oral toxicity studies with the strain of rats used in this study (rats were about 19 weeks old at the time of blood sampling). Relative changes in mean values compared to the concurrent control group are indicated between parentheses.
• Lower haemoglobin from 5 mg/kg bw/day onward (16%, 17% and 8% at 5, 15 and 50 mg/kg bw/day, respectively; latter difference not statistically significant).
• Lower mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) from 5 mg/kg bw/day onward (9% for MCH, 5-8% for MCHC).
• Lower mean values for the number of red blood cells and haematocrit at 5 and 15 mg/kg bw/day (about 10%, not statistically significant).
• Higher percentage of reticulocytes at 5 and 15 mg/kg bw/day (60% and 130%, respectively). The value in one 15 mg/kg bw/day female was about five-fold higher compared to control values.
• Higher red blood cell distribution width (RDW) at 5 mg/kg bw/day (135%).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Females:
• Higher alkaline phosphatase (ALP) at 50 mg/kg bw/day (69%).
• Lower albumin at 15 and 50 mg/kg bw/day (7 and 14%, respectively).
• Lower total protein at 15 and 50 mg/kg bw/day (7% at both dose levels).
• Lower creatinine from 5 mg/kg bw/day onward (17% at 5 mg/kg bw/day, 20% at the higher dose levels).
The following statistically significant differences between treated females and concurrent controls were considered to be due to the difference in physiological status: lower values for alanine aminotransferase (ALAT) activity, cholesterol, bile acids, urea and inorganic phosphate, and higher values for sodium and chloride.
The other differences noted in treated females were considered unrelated to treatment due to somewhat high concurrent control values (aspartate aminotransferase (ASAT) activity) or because they occurred in the absence of a dose-related trend (lower ALP and higher fasting glucose at 5 mg/kg bw/day).

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Females: Statistically significantly lower mean values for hind limb grip strength were noted at 15 and 50 mg/kg bw/day (relative differences from controls: about 30-40%). Values in treated animals remained in the normal historical range for rats of this strain and age, except for those of two females in Group 3 and two females in Group 4 which were below this range.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
The lower motor activity noted in females treated at 5 mg/kg bw/day, particularly from test interval 4 onwards (i.e. >15 minutes), occurred in the absence of a dose-related trend and was therefore considered to be unrelated to treatment. One female at 15 mg/kg bw/day showed low motor activity throughout the 1-hour test period. This might be related to her health condition (she showed piloerection and had an abnormal pregnancy). It was noted that two other 15 mg/kg bw/day females and several 50 mg/kg bw/day females with piloerection and abnormal pregnancies showed normal motor activity. This female had normal outcomes for other measures in the neuromuscular domain (including gait, air righting reflex and grip strength). Therefore, her lower motor activity was considered not to represent a direct effect of the test item on this neuromuscular endpoint.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Females:
There were statistically significant organ weight changes at 15 and 50 mg/kg bw/day in liver (decreased absolute and relative weight) and thymus (increased relative weight). These organ weight changes were regarded to be related to the different physiologic state of the test item-treated females compared to the controls: lactating females show a decreased thymus weight and increased liver weight compared to non-lactating females. In the current study all females of the control group were lactating, compared to none of the test item-treated groups. There were no microscopic correlates to these weight changes. Therefore these weight changes in liver and thymus of females were regarded to be unrelated to the treatment with the test item.
Other organ weight changes of note were present in females at 5 mg/kg bw/day and consisted of an increased relative spleen weight (microscopic correlate: increased hematopoiesis) and increased absolute and relative ovary and uterus weight (considered correlating to the time of necropsy: shortly after delivery of the pups). To a lesser extent, the weights of the ovaries and uterus were also increased in females at 15 and 50 mg/kg bw/day, which was considered to be related to their physiologic state (not pregnant, implantations only or suspected total litter loss).
The remaining statistically significant organ weight changes in females were in line with the lower terminal body weight (kidneys, brain, adrenals) or unrelated to treatment due to the lack of a dose-related pattern (thyroid gland).

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related macroscopic findings in female rats.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

est item-related microscopic findings were observed in the thymus, thyroid gland, liver, spleen, and female reproductive organs, as described below.
• Thymus: Increased apoptosis of lymphocytes was recorded in females starting at 5 mg/kg bw/day.
• Thyroid gland: An increased incidence and severity of hypertrophy of follicular cells was recorded in females at 50 mg/kg bw/day.
• Spleen: An increased incidence and severity of extramedullary hematopoiesis was recorded in females at 50 mg/kg bw/day.
The high incidence and severity of extramedullary hematopoiesis in the spleen (and liver) of females at 5 and 15 mg/kg bw/day was considered to be mainly related to blood loss, due to abnormal pregnancies.
• Uterus: Vascular necrosis at the implantation sites was recorded in six females at 5 mg/kg bw/day and one female at 15 mg/kg bw/day.
Hemorrhage in the uterine lumen and/or implantation sites was recorded in seven females at 5 mg/kg bw/day and three females at 15 mg/kg bw/day. The hemorrhage was considered to be related to the vascular necrosis and/or recent parturition.
There were no other test item-related histologic changes. 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):

Estrous cycle:
Treatment at 50 mg/kg bw/day resulted in abnormal estrous cycles in 7/10 females: one female (not pregnant) had an irregular cycle (persistent di-estrus); six females (not pregnant, implantations only or suspected total litter loss) had an acyclic cycle (at least 10 days without estrus). The remaining three females (all not pregnant) had regular cycles of 4 days.
At 15 mg/kg bw/day, one female (suspected total litter loss) had an irregular cycle (persistent di-estrus). An irregular cycle occasionally occurs at low incidence in untreated controls. However, taken in the context of the cycling abnormalities at 50 mg/kg bw/day, it cannot be excluded that the irregular cycle in this female was related to treatment.
Length and regularity of the estrous cycle were not affected at 5 mg/kg bw/day. All 5 mg/kg bw/day females had regular cycles of 4 days.

Details on results:

All test item treated females failed to deliver healthy pups and were sacrificed about 1 to 2 weeks before the control females.
Clinical signs of toxicity occurred in treated females towards the end of the post-coitum period. These included piloerection in one female at 5 mg/kg bw/day and most females at 15 and 50 mg/kg bw/day and hunched posture in a few females at the higher dose levels. It cannot be excluded that these clinical signs, at least in part, were secondary to pregnancy difficulties.
Functional observations showed lower mean values for hind limb grip strength at 15 and 50 mg/kg bw/day. As there were no corroborative clinical signs or changes in other measures in the neuromuscular domain (including forelimb grip strength, gait, air righting reflex and motor activity), these differences in hind limb grip strength were considered not to be adverse.
Body weight gain was reduced at 15 and 50 mg/kg bw/day throughout the treatment period, and at 5 mg/kg bw/day during the first two weeks of treatment. Females at 15 and 50 mg/kg bw/day initially had only slightly lower mean body weights (up to about 5%), but between Days 11-20 of the post-coitum period, 4-9% body weight loss occurred in all non-pregnant females in the 15 mg/kg bw/day (n=3) and 50 mg/kg bw/day (n=6) groups . In addition, the two females in the 50 mg/kg bw/day group with implantation sites only lost 10% and 9% of their body weight from Days 14-20 post-coitum and Days 17-20 post-coitum, respectively. This weight loss was considered adverse. Females at 15 and 50 mg/kg bw/day consumed less food than controls during the post-coitum period. A possible relation with the observed aborted pregnancies and/or pup mortality cannot be excluded.
Changes in haematology parameters were noted in females at all dose levels. Total white blood cell count was lower in all treated groups. In the absence of morphological correlates,
this change was likely to be non-adverse. The red blood cell changes in females at 5 and 15 mg/kg bw/day (lower haemoglobin, haematocrit, red blood cell count, MCH and MCHC, higher percentage of reticulocytes and red blood cell distribution width), accompanied by extramedullary hematopoiesis in spleen and liver, were considered to be mainly related to blood loss due to abnormal pregnancies. The decreases in MCH and MCHC in females at 50 mg/kg bw/day were considered non-adverse as they occurred in the absence of decreases in haemoglobin, red blood cell count and haematocrit.
Clinical biochemistry changes consisted of higher alkaline phosphatase (ALP) activity and lower albumin and total protein starting at 15 mg/kg bw/day and lower creatinine starting at 5 mg/kg bw/day. Based on their modest magnitude (ALP less than two-fold, total protein less than 10%, creatinine approximately 20% in females) and/or absence of morphologic correlates, these changes were likely to be nonadverse.
Non-adverse, treatment-related microscopic changes were observed in the thymus (increased apoptosis of lymphocytes from 5 mg/kg bw/day) and spleen (increased extramedullary haematopoiesis at 50 mg/kg bw/day). Based on their low severity and the absence of accompanying degenerative or inflammatory changes, these findings were regarded as non-adverse.
Adverse morphological changes in parental reproductive organs were observed from 5 mg/kg bw/day onward:
Microscopic changes in the uterus consisted of vascular necrosis of arteries at the implantation sites and hemorrhage in the uterine lumen and/or implantation sites in most females at 5 mg/kg bw/day and a few females at 15 mg/kg bw/day. These morphological changes may at least in part be the underlying cause of the aborted pregnancies and pup mortality observed in this study.
Other treatment-related adverse changes in reproductive parameters included abnormalities in estrous cyclicity (1/10 female at 15 mg/kg bw/day and 7/10 females at 50 mg/kg bw/day), and decreases in the number of implantation sites and fertility index at 15 and 50 mg/kg bw/day (respectively, 7/10 and 4/10 females with evidence of mating were pregnant).
For further details on general and reproductive toxicity see sections 7.5.1 and 7.8.1 of this IUCLID (cross-reference).

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:

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 43% at 5 mg/kg bw/day. On an individual animal level, post-implantation survival was normal (no or only one or two implantations lost) in 3/10 pregnant 5 mg/kg bw/day females, and reduced (6-10 implantations lost) in the remaining four 5 mg/kg bw/day females with offspring. 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. This explains why no pups were recorded for the three 5 mg/kg bw/day females with normal numbers of implantation sites, normal gestational body weight gain and normal weight loss after parturition which had (suspected) total litter loss before first litter check.
At the higher dose levels (15 and 50 mg/kg bw/day), pups were cannibalized prior to first litter check. Therefore, post-implantation survival index was 0%.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

In the 5 mg/kg bw/day group only 4/10 pregnant females had living pups at first litter check, but these pups had to be euthanized due to poor condition on the same day (PND 1). Three females had only dead pups at first litter check. The remaining three females had no pups that could be evaluated: pups of dam 58 were cannibalized before first litter check, and for two females total litter loss was suspected based on available body weight data.
At the higher dose levels, 7/10 females at 15 mg/kg bw/day and 2/10 females at 50 mg/kg bw/day 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: All females had implantation sites in their uterus. 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 normal pregnancy. The body weight loss recorded in these females after Day 20
post-coitum is indicative for a delivery had taken place. The remaining two pregnant females in the 50 mg/kg bw/day group had implantation sites only.

Changes in pregnancy duration:

effects observed, treatment-related

Description (incidence and severity):

See above.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Duration of gestation was normal in all seven 5 mg/kg bw/day females that delivered (mean value of 22.0 days compared to 21.6 days in concurrent controls).
At the higher dose levels, 7/10 females at 15 mg/kg bw/day and 2/10 females at 50 mg/kg bw/day 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: All females had implantation sites in their uterus. 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 normal pregnancy. The body weight loss recorded in these females after Day 20 post-coitum is indicative for a delivery had taken place. The remaining two pregnant females in the 50 mg/kg bw/day group had implantation sites only. As in the 15 and 50 mg/kg bw/day groups delivery of pups was not recordable, a gestation index of 0% was derived and duration of gestation could not be determined.

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

Fertility index was reduced to 70% (7 pregnant females) at 15 mg/kg bw/day and 40% (4 pregnant females of which two females had implantation sites only) at 50 mg/kg bw/day.
Fertility index was unaffected by treatment at 5 mg/kg bw/day (100%).

Other effects:

no effects observed

Description (incidence and severity):

Mating index was not affected by treatment. All females showed evidence of mating (mating index 100%). Precoital time was considered not to be affected by treatment. 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.

Details on maternal toxic effects:

Adverse morphological changes in parental reproductive organs were observed from 5 mg/kg bw/day onward. Microscopic changes in the uterus consisted of vascular necrosis of arteries at the implantation sites and hemorrhage in the uterine lumen and/or implantation sites in most females at 5 mg/kg bw/day and a few females at 15 mg/kg bw/day These morphological changes may at least in part be the underlying cause of the aborted pregnancies and pup mortality observed in this study.
Other treatment-related adverse changes in reproductive parameters included abnormalities in estrous cyclicity (1/10 female at 15 mg/kg bw/day and 7/10 females at 50 mg/kg bw/day), and decreases in
the number of implantation sites and fertility index at 15 and 50 mg/kg bw/day (respectively, 7/10 and 4/10 females with evidence of mating were pregnant).
At 15 and 50 mg/kg bw/day, all females that delivered or were suspected to have delivered had lost their pups before a first litter check could be performed. Consequently, post-implantation survival and gestation indices were 0%.

Key result

Dose descriptor:

LOAEL

Remarks:

maternal toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

changes in pregnancy duration

dead fetuses

histopathology: non-neoplastic

pre and post implantation loss

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

uterus

Description (incidence and severity):

Vascular necrosis of arteries at the implantation sites and hemorrhage in the uterine lumen and/or implantation sites starting at 5 mg/kg bw/day

Fetal body weight changes:

not examined

Description (incidence and severity):

No fetal examinations were performed, as females were allowed to litter normally.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): No fetal examinations were performed, as females were allowed to litter naturally.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

At 5 mg/kg, live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was reduced to 18%. On an individual litter level, four litters included 1-3 living pups (and 3-9 dead pups) and three litters included only dead pups (3, 5 and 9 dead pups, respectively). The remaining three females had (suspected) total litter loss before first litter check.
At the higher dose levels of 15 and 50 mg/kg bw/day, respectively 7/10 and 2/10 females were suspected to have delivered based on available body weight data. However, as pups were cannibalized before first litter check, no live birth index could be determined.

Changes in sex ratio:

not specified

Description (incidence and severity):

Sex ratio at PND 1 appeared unaffected by treatment at 5 mg/kg bw/day as indicated by the approximately equal proportions of males and females for both the living pup (5 males, 4 females) and the dead pups (21 males, 19 females).
At the higher dose levels (15 and 50 mg/kg bw/day) sex ratio could not be evaluated due to the lack of offspring.

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 markedly reduced at 5 mg/kg bw/day (1.3 versus 11.3 in controls). Four of the seven dams had 1-3 living pups at first litter check, the remaining three dams had only dead pups.
At the higher dose levels (15 and 50 mg/kg bw/day), all pups were cannibalized prior to first litter check.
In pups, at 5 mg/kg bw/day reduced pup body weight at birth (combined body weight for male and female pups) was 12% lower than in the concurrent control group. The relative differences were 12% in male and 16% in female pups. At higher dose levels no pups were observed at the first litter check.

Changes in postnatal survival:

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 43% at 5 mg/kg bw/day. On an individual animal level, post-implantation survival was normal (no or only one or two implantations lost) in 3/10 pregnant 5 mg/kg bw/day females, and reduced (6-10 implantations lost) in the remaining four 5 mg/kg bw/day females with offspring. 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. This explains why no pups were recorded for the three 5 mg/kg bw/day females with normal numbers of implantation sites, normal gestational body weight gain and normal weight loss after parturition, which had (suspected) total litter loss before first litter check).
At the higher dose levels (15 and 50 mg/kg bw/day), pups were cannibalized prior to first litter check.
Therefore, post-implantation survival index was 0%.

External malformations:

not specified

Description (incidence and severity):

No external abnormalities were noted in 5 mg/kg bw/day pups. There were no pups at the higher dose levels (15 an 50 mg/kg bw/day) available for macroscopic examination.

Skeletal malformations:

not examined

Description (incidence and severity):

No detailed examinations were performed, but no macroscopic anomalies were noted in 5 mg/kg bw/day pups. There were no pups at the higher dose levels (15 an 50 mg/kg bw/day) available for macroscopic examination.

Visceral malformations:

not examined

Description (incidence and severity):

No detailed examinations were performed, but no macroscopic anomalies were noted in 5 mg/kg bw/day pups. There were no pups at the higher dose levels (15 an 50 mg/kg bw/day) available for macroscopic examination.

Other effects:

not specified

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups appeared unaffected by treatment at 5 mg/kg bw/day. Note: only four litters (with only one or two pups/sex per litter) were available for evaluation. At the higher dose levels (15 and 50 mg/kg bw/day) anogenital distance could not be evaluated due to the lack of offspring.
No PND 13 pups were available for determination of areola/nipple retention and serum T4 levels in the treated groups.
All living pups in the 5 mg/kg bw/day group (9 in total) were lethargic, cold, and/or had no or less milk in the stomach at first litter check. They were therefore sacrificed at PND 1. Macroscopic findings among the 5 mg/kg bw/day pups that were found dead at first litter check included absence of milk in the stomach, cannibalism and beginning autolysis. There were no pups at the higher dose levels (15 an 50 mg/kg bw/day) available for macroscopic examination.

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in litter size and weights

other: Reduced post-implantation survival to 43% at the lowest dose level and to 0% at higher dose levels

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

The concentrations analyzed in the formulations of the 5, 15 and 50 mg/kg bw/day treatment groups were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%; actual range: 97-102%, n = 6 for the 5 and 50 mg/kg bw/day treatment groups, n=2 for the 15 mg/kg bw/day treatment group) and prepared homogenously (i.e. coefficient of variation ≤ 10%; actual range: 1.2-6.1%), except for those of the Group 2 prepared and analyzed on treatment day 2 (mean accuracy of 83%). This lower accuracy was attributed to inhomogeneity of the formulation (coefficient of variation was 30%). To facilitate the homogenization process, it was decided to keep all formulations for a longer period on the magnetic stirrer. Formulations prepared according to this revised protocol were used first from treatment Day 4 onward and both accuracy and homogeneity were confirmed (coefficient of variation 6.1%, mean accuracy 97% (n = 6)).

The formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 6 hours (mean accuracy of 86% and 101% for the 5 and 50 mg/kg bw/day, respectively; n = 2 for both groups).

Conclusions:

In a GLP-compliant OECD guideline 422 study with rats treated by gavage, adverse test item-related effects in uterus (vascular necrosis of arteries at the implantation sites and hemorrhage in the uterine lumen and/or implantation sites) were observed at the lowest dose level of 5 mg/kg bw/day in maternal animals. None of the test item-treated animals had healthy offspring. Only 4/10 females at the lowest dose level of 5 ppm had live pups; however, they were sacrificed in extremis at PND1. At higher dose levels, all pups were either cannibilized before the first litter check, or only the implantation sites were observed in treated females that were pregnant. The viability index was 0% at all dose levels. Post-implantation survival index was reduced to 43% at 5 mg/kg bw/day and to 0% at higher dose levels. Litter size was markedly reduced at 5 mg/kg bw/day (1.3 vs. 11.3 in controls). Live birth index was reduced to 18% at 5 mg/kg bw/day and to 0% at higher dose levels. Based on these effects, the lowest dose level of 5 mg/kg bw/day was considered to be a LOAEL for maternal and developmental toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

5 mg/kg bw/day

Study duration:

subacute

Species:

rat

Justification for classification or non-classification

A classification and labelling of this substance as Repr Cat 1B is warrranted based on the findings in the OECD 422.