reaction mass of mixed (3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl) phosphates, ammonium salt

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 September 2016 - 12 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

yes

Remarks:

The study was conducted in mice instead of rats.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (18°C to 24°C)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stable in water over the range of concentrations used in the current study for at least 5 or 10 days at room temperature

Test animals

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

The animal model, the CD-1 mouse, is recognized as appropriate for reproductive toxicity studies and has been proven to be susceptible to the effects of reproductive toxicants. In addition, Charles River Ashland has reproductive historical control data in the Crl:CD(ICR) mouse.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, USA
- Females nulliparous and non-pregnant: not applicable, reproductive/developmental screening study
- Age at study initiation: ca. 35 days upon receipt, ca. 6 weeks at study day 0.
- Weight at study initiation: main study: males: 20.7 g to 32.5 g, females: 19.2 g to 25.4 g, clinical pathology group: males 23.3-32.5 g, females 19.2-24.9 g
- Fasting period before study: none
- Housing:
Upon receipt: 2–3 mice/cage by sex in clean, solid-bottom cages with bedding material, for 3 days
Thereafter and until pairing: individually in clean, solid-bottom cages with bedding material
During mating: in the home cage of the male
After mating: males: individually until scheduled necropsy
Females that delivered: individually housed until euthanasia on Lactation Day 21
Females that failed to deliver: individually housed until Postmating Day 23
Females that were not mated and clinical pathology animals: indvidually in clean solid-bottom cages with bedding material until euthanasia
F1 pups: after weaning together by litter in solid-bottom cages with bedding material until PND 28, after PND28 individually until euthanasia.
Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, ad libitum
- Water: municipal water, ad libitum
- Acclimation period: 9 days

DETAILS OF FOOD AND WATER QUALITY: appropriate analyses of the diet performed by the manufacturer and provided to Charles River. Municipal water supplying the facility was sampled for contaminants according to Charles River SOPs. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.9 to 21.9
- Humidity (%): 30.4% to 61.2%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 September 2016 To: 12 March 2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance formulations were prepared daily during 29 Sep 2016 to 02 Nov 2016 and then approximately weekly for the remainder of the study. The test substance formulations were prepared as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature (18°C to 24°C). The test substance formulations were stirred continuously throughout the preparation,
sampling, and dose administration procedures. The first test substance dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

VEHICLE
- Concentration in vehicle: 0, 0.1, 2 and 10 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until mating, for a maximum of 14 days
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- Further matings if unsuccessful: no
- After successful mating each pregnant female was caged individually in clean, solid-bottom cages with bedding material

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance formulations were expected to be solutions in deionized water and stable over the range of concentrations used in the current study for at least 5 or 10 days at room temperature. Therefore stability and homogeneity of the test substance formulations were not assessed on this study. However, solubility and stability were not established at the time of the first preparation for use on study. Therefore, samples were collected from the top, middle, and bottom strata of the first 0.1, 2, and 10 mg/mL dosing formulations and from the middle stratum of the first control dosing formulation for concentration analysis and possible future homogeneity determination. Samples for concentration analysis were also collected from the middle stratum of each dosing formulation (including the control group) prepared during the fourth, ninth, and last weeks of the study. One set of samples from each collection was analyzed. All remaining samples were stored at room temperature (18°C to 24°C) as back-up. All analyses were conducted by the Charles River Analytical Chemistry Department using a validated high performance liquid chromatography method using tandem mass spectrometry detection.

Duration of treatment / exposure:

Males: 90 days prior to mating, throughout the mating and until 1 day prior to euthanisia, for a total of 109-113 days
Females: 90 days prior to mating, during mating and through lactation day 20, for a total of 130-142 days
Females that failed to deliver: 90 doses prior to mating, during mating and through postmating day 23 for a total of 113 days
Females that were not mated: 109 days (euthanized together with males)
Clinical pathology animals: 75 consecutive days
F1 animals: 21 days post-weaning (PND22-42).

Frequency of treatment:

Once daily for 7 days/week, approximately at the same time each day.

Details on study schedule:

- Age at mating of the mated animals in the study: 19 weeks (F0 generation)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Concurrent vehicle controls

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

Main study animals: 20/sex/dose
Additional 5 females in the control and high-dose group were not mated, but were administered the test substance throughout the study and were utilized for gender comparison without gestation influence.
Clinical pathology phase animals: 15/sex/dose
F1 animals after culling: 16-20/sex/group (1/sex/litter)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dosage levels were selected based on available pharmacokinetic studies and repeated dose studies with this test substance or analogous substances. Within this class of chemistry, there is a substantial amount of data on the six carbon acid and the eight carbon acid, but very little on this test substance (the seven carbon acid). In general, the most notable effect of perfluorinated carboxylic acids in mice are enlarged livers due to activation of the PPAR alpha receptor. The potency of this activation appears to be proportional to chain length, with eight carbons and above causing substantially more activation, and therefore enlargement, than perfluorinated carboxylic acids with less than 8 carbons. Publications suggest that the potency of the eight carbon acid is approximately 1 order of magnitude higher than the six or seven carbon acid. The seven carbon acid is more potent than the six carbon acid, but only approximately 2 to 5 times.
The elimination half-life of the perfluorinated carboxylic acids varies with chain length, with eight carbon and longer chains being eliminated more slowly than the six carbon and shorter chains. Available pharmacokinetic data with this test substance (the seven carbon acid) is more similar to the six carbon than the eight, with an estimated half-life of between 2 and 4 hours.
The high-dosage level of 50 mg/kg bw/day was high enough to cause enlarged livers in the male mice, and likely in the female mice, which may result in effects in reproductive endpoints. The mid-dosage level of 10 mg/kg bw/day was expected to show no or minimal liver enlargement in male mice with no liver enlargement expected in the females. The reproductive endpoints were not likely to be affected at the mid-dosage level, but there was some uncertainty. Because of this uncertainty, a low-dosage level of 0.5 mg/kg bw/day was selected. This dosage level was expected to be the NOAEL for all endpoints examined in this study.

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and moribundity, and also for signs of toxicity ca. 1 hour after dosing. Females expected to deliver were observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: individual clinical examinations were recorded daily; detailed clinical examinations were conducted weekly prior to dose administration during the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: main study males: weekly throughout the study and prior to the scheduled euthanasia. Main study females: weekly untl evidence of copulation or until euthanasia (non-mated females); after evidence of mating on Gestation days 0, 4, 7, 11, 14, and 18 and on Lactation Days 0 (when possible), 1, 4, 7, 10, 14, 17, and 21. Clinical pathology animals: weekly throughout the study until euthanasia

FOOD CONSUMPTION: yes
- Time schedule for examinations: main study animals: weekly on the corresponding body weight days until the start of the mating period; following mating for mated females food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, and 18 and on Lactation Days 1, 4, 7, 10, 14, 17, and 21. Clinical pathology animals: weekly beginning on the first day of dose administration until euthanasia.

- FOOD INTAKE: no

WATER CONSUMPTION: no

OTHER:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to randomization and near the end of the dosing period (study week 15) for males and non-mated females; for mated females during study week 17).
- Dose groups that were examined: all main study animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: main study animals: at the scheduled necropsy (study week 15 for males and non-mated females; lactation day 21 for mated females); clinical pathology animals: at scheduled necropsy (day 75).
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: No
- How many animals: main study: 10/sex/dose (out of a total of 15 animals/sex/dose 3 separate groups of 5 mice/sex/dose were used for hematology, coagulation and clinical chemistry evaluation) + 5 non-mated females in high dose and control groups; clinical pathology animals: 10/sex/dose (out of a total of 15 animals/sex/dose 3 separate groups of 5 mice/sex/dose were used for hematology, coagulation and clinical chemistry evaluation).
- Parameters examined: WBC, RBC, hemoglobin, hematocrit, MCV, MCH, MCHC, platelet counts, PT, APTT, reticulocyte count percent (RETIC), RETIC absolute; differential leukocyte count (neutrophils, lympholytes, monocytes, eosinophils, basophils, large unstained cells); RDW; hemoglobin distribution width (HDW), platelet estimated, RBC morphology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: main study animals: at the scheduled necropsy (study week 15 for males and non-mated females; lactation day 21 for mated females); clinical pathology animals: at scheduled necropsy (day 75).
- Animals fasted: no
- How many animals: main study: 5/sex/dose (out of a total of 15 animals/sex/dose 3 separate groups of 5 mice/sex/dose were used for hematology, coagulation, and serum chemistry collection) + 5 non-mated females in high dose and control groups; clinical pathology animals: 5/sex/dose (out of a total of 15 animals/sex/dose 3 separate groups of 5 mice/sex/dose were used for hematology, coagulation, and serum chemistry collection).
- Parameters examined: albumin, total protein, globulin (by calculation); albumin/globulin ratio (by calculation); total bilirubin, urea nitrogen, creatinine, alkaline phosphatase, ALP, ALAT, ASAT, gamma glutamyltransferase (GGT), glucose, total cholesterol, calcium, chloride, phosphorus, potassium, sodium, sorbitol dehydrogenase, triglyceride, bile acids, appearance (degree of hemolysis, lipemia, and icterus)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males: study week 15 (last week of test substance administration); mated females: lactation day 21.
- Dose groups that were examined: all main animals, on 10 animals/sex/group
- Battery of functions tested: home cage observations, handling observations, open field observations, sensory activity (approach response, pupil response, forelimb extension, air righting reflex, touch response, tail poinch response, eyeblink response, hindlimb response, olfactory orientation), grip strength (hindlimb extensor strengh, hindlimb foot splay, grip strengh-hind and forelimb, rotarod performance), motor actvity (total movements and ambulations).

THYROID HORMONE ANALYSIS: yes
Blood samples were collected from main study animals for thyroid hormone analysis immediately prior to euthanasia (females that delivered: lactation day 21, females that failed to deliver: postmating day 23); only male samples were analysed.

Oestrous cyclicity (parental animals):

Oestrous cycle (main study animals): vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the estrous cycle of each female selected for mating for 14 days prior to mating and continuing until evidence of copulation was observed or until termination of the mating period for females with no evidence of mating. The average cycle length was calculated and reported for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] until the detection of evidence of mating), beginning with the first day of dose administration. Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the mean individual estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight, PAS staining of testes and epididymides

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter ([4]/sex/litter as nearly as possible); standardization of litter size was not performed on litters with fewer than 8 pups.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
During weaning: number and sex of pups (PND 0, 4 and 21), stillbirths, live births, postnatal mortality, body weights (measured on PND 1, 4, 7, 10, 14, 17 and 21), clinical signs, physical or behavioural abnormalities, anogenital distance (PND1), presence of nipples/areolae in male pups (PND 13).

Post-weaning, the following observations were performed:
Clinical signs observations: twice daily for moribundity and mortality, individual clinical observations recorded daily. All animals were also observed for signs of toxicity approximately 1 hour following dose administration.
Detailed clinical examinations: weekly (prior to dose administration)
Body weights: weekly beginning on PND22.
Food consumption: weekly beginning on PND28 (following individual housing).
Balanopreputial separation: all males starting from PND 27, daily. Body weights were recorded when balanopreputial separation was achieved.
Vaginal patency: all females starting from PND 21, daily. Body weights were recorded when vaginal patency was achieved (vaginal lumen first observed).

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities. A detailed gross necropsy was performed on any pup found dead or euthanized due to death of dam after PND 4. Intact offspring that were found dead from PND 0 to 4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. Possible cause of death was determined for pups born or found dead.
On PND21 pups not selected for F1 generation were euthanized. Gross necropsy was conducted on all non-selected pups. From 1 pup/sex/litter blood was collected for thyroid hormone determination, and the thyroids (with parathyroids, if present) were weighed (following fixation) and placed in 10% neutral-buffered formalin for possible histopathological examination. In addition, two female pups/litter, when possible, were euthanized by carbon dioxide inhalation and selected for collection of mammary glands for whole mount histopathology on PND 21.

THYROID HORMONE ANALYSIS:
On PND 4: Blood samples for possible future thyroid hormone analysis were collected from 2 culled pups/litter (pooled by litter) on PND 4. The samples were stored but not analysed. Remaining culled pups (not used for blood collection) were weighed, euthanized by an intraperitoneal injection of sodium pentobarbital on PND 4, and discarded.
On PND 21: On PND 21, 1 pup/sex/litter was euthanized by exsanguination and blood samples were collected for thyroid hormone analysis immediately prior to euthanasia.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: not performed.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: not performed.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on day 109-113
- Females that delivered: following 90 days treatment, mating and lactation until day 20 (euthanasia on day 130-142)
- Females that failed to deliver: following 90-day treatment, mating and until postmating day 23 (euthanasia on day 113)
- Non-mated females: the same as males
- Females with total litter loss: within 24 hours of litter loss
- Clinical pathology animals: after 75 days

GROSS NECROPSY
- Gross necropsy was performed on all main study animals and all clinical pathology phase animals. Necropsy included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera.


HISTOPATHOLOGY / ORGAN WEIGHTS
Histopahology was performed on all main study animals and all animals found dead or euthanized in extremis. In addition, gross lesions from all animals in all groups and the liver from F0 males and females in the 0.5 and 10 mg/kg/day groups were examined. The following tissues were preserved and examined by pathologist: adrenal glands, aorta, bone with marrow (sternebrae, femur), brain, Cowper's gland, coagulating glands, eyes with optic nerve, gallbladder, gastrointestinal tract, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, Peyer's patches, glans penis, heart, kidneys, liver, lungs including bronchi, LABC muscle complex, lymphn nodes (axiliary, mandibular, mesenteric), ovaries and oviducts, pancreas, sciatic nerve, pituitary gland, prostate, salivary gland, seminal vesicles, skeletal muscle, skin with mammary gland, spinal cord, spleen, testes with epididymides and vas deference, thymus, thyroids with parathyroids, tongue, trachea, urinary bladder, uterus with cervis and vagina.

The following organ weights were recorded in main study animals: adrenal glands, brain, Cowper's gland, epididymides, glans penis, heart, kidneys, LABC muscle complex, liver, ovaries with oviducts, pituitary gland, seminal vesicle with coagulating gland and fluid, spleen, testes, thymus gland, thyroids with parathyroids, uterus.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed on PND42 (21 days post-weaning).
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
Microscopic examination was performed on the liver and kidneys from all animals in all groups, the adrenal glands and spleen from all females in all groups, and the mammary gland from all females in the control and 50 mg/kg bw/day groups at the scheduled necropsies. The mammary glands collected from nonselected females on PND 21 in the control and 50 mg/kg bw/day groups were also examined microscopically.

The following organs were weighed from all F1 animals: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries with oviducts, spleen, testes, thymus gland, uterus.

Statistics:

Parental mating, fertility, conception, and copulation indices were analyzed using the Chi-square test with Yates’ correction factor. Parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, estrous cycle length, precoital intervals, gestation length, numbers of former implantation sites, and unaccounted-for sites, number of pups born, live litter size on PND 0, absolute and relative organ weights, clinical pathology values, anogenital distance (absolute and relative to the cube root of body weight), number of nipples/areolae, and FOB data values were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's testwas used to compare the test substance-treated groups to the control group. FOB parameters that yield scalar or descriptive data in the test substance-treated groups were compared to the control group using Fisher’s Exact test. Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA to determine intergroup differences. Ifthe nonparametric ANOVA revealed significant (p < 0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group.

Reproductive indices:

The following indices were calculated:
Mating index (%) (male/female) = 100% x (number of males/females with evidence of mating/confirmed pregnant)/(total number of males/females used for mating)
Male fertility index (%) = 100% x (number of males siring a litter/total number of males used for mating)
Male copulation index (%) = 100% x (number of males siring a litter/number of males with evidence of mating (or females with confirmed pregnancy)
Female fertility index (%) = 100% x (number of females with confirmed pregnancy/total number of females used for mating)
Females conception index (%) = 100% x (number of females with confirmed pregnancy/number of females with evidence of mating)

Offspring viability indices:

The following offspring indices were calculated:
Mean live litter size = (total number of viable pups on PND0)/(number of litters with viable pups on PND 0)
Postnatal survival between birth and PND 0 or PND 4 (% per litter) = 100% x ((sum of viable pups per litter on PND0 or PND4/number of pups born per litter)/number of litters per group)
Postnatal survival for all other intervals (% per litter) = 100% x ((sum of viable pups per olitter at the end of interval N/viable pups per litter at the start of period N)/number of litters per group),
where N = PND 0–1, 1–4 (pre-selection), 4 (post-selection)–7, 7–10, 10–14, 14–17, 17–21, birth to 4 (pre-selection), and 4 (post-selection)–21.
Pups that were euthanized due to death of the dam were excluded from pup viability calculations.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

Main study animals: no test substance-related clinical observations were noted for F0 males and females at 0.5, 10, and 50 mg/kg bw/day. Clinical observations noted in the test substance-treated groups at the daily examinations, detailed physical examinations, and approximately 1 hour following dose administration, including hair loss and/or scabbing on various body surfaces, were noted infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Main study animals:
There were no test substance-related effects on survival at 0.5, 10, and 50 mg/kg bw/day. In the 50 mg/kg bw/day group, one female was found dead on Study Day 12; no significant clinical observations were noted for this female. At necropsy, an advanced degree of autolysis precluded a complete examination. There were no macroscopic or microscopic findings and the cause of death could not be determined for this animal. Because there were no adverse observations that correlated to other females within this dose group, this death was not considered test substance-related.
One male in the 0.5 mg/kg bw/day group was found dead on Study Day 103 after being noted with a pale body and hypoactivity approximately 30 minutes prior to death and approximately 1.5 hours following dose administration; there were no macroscopic or microscopic findings and the cause of death could not be determined. Because there were no test substance-related deaths in the higher dose levels, this death was not considered test substance-related.
One male in the 10 mg/kg bw/day group was euthanized in extremis on Study Day 26 following clinical observations of hypoactivity, cool and pale body, and yellow material on the urogenital area on the day of euthanasia at the daily examination and severe body
weight loss (14.9%) and reduced food consumption (2.9 g feed/day) during Study Days 14–21. At necropsy, this male was noted with a thickened thymus and a subcutaneous mass in the left axilla. The subcutaneous mass correlated microscopically to acute inflammation and was considered the cause of moribundity. Moderate degeneration of the muscle of the esophagus was noted. Additional microscopic findings that were likely secondary to the moribund state included myeloid hyperplasia of the sternal and femoral bone marrow, increased extramedullary hematopoiesis of the spleen, and lymphoid depletion of the thymus. Based on the lack of a dose response, this death was not considered test substance-related.
In the control group, 1 female was found dead on Lactation Day 15. There were no macroscopic findings for this female and microscopic findings were limited to minimally increased extramedullary hematopoiesis of the spleen and moderate unilateral periocular hemorrhage; the cause of death could not be determined. All other F0 males and females survived to the scheduled necropsies.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Main study animals: mean body weights and body weight gains in the 0.5, 10, and 50 mg/kg bw/day group F0 males were unaffected by test substance administration throughout the study. None of the differences from the control group were statistically significant. In females, a significantly (p < 0.05) higher mean body weight gain was noted in the 0.5 mg/kg bw/day group compared to the control group during Study Days 21–28, and a significantly (p < 0.01) lower mean body weight gain was noted in the 50 mg/kg bw/day group compared to the control group during Lactation Days 1–4. These differences were transient and not of sufficient magnitude to affect the mean body weights at these dosage levels, and therefore were not considered test substance-related. No other effects on the body weights or body weight gains were observed in females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Main study animals:
Males: mean food consumption, evaluated as g/animal/day and g/kg bw/day, in the 0.5, 10, and 50 mg/kg bw/day group males was similar to that in the control group throughout the study. No statistically significant differences were observed, with the following exception. Significantly (p < 0.05) lower mean food consumption (g/kg bw/day value only) was noted in the 50 mg/kg bw/day group compared to the control group during Study Days 56–63; this difference was transient and not of sufficient magnitude to affect mean body weights at this dosage level, and therefore was not considered test substance-related.
Females: no effects on mean food consumption were observed during the pre-mating, mating and gestation periods. During lactation, test substance-related, lower mean maternal food consumption, evaluated as g/animal/day and g/kg bw/day, was noted in the 50 mg/kg bw/day group compared to the control group generally throughout lactation and resulted in lower mean food consumption in this group when the entire lactation treatment period (Lactation Days 1–21) was evaluated; differences were generally significant (p < 0.01). However, this was likely secondary to the decreased nutritional demand of the smaller pups and considered nonadverse. Mean maternal food consumption in the 0.5 and 10 mg/kg bw/day groups was unaffected by test substance administration during lactation. Differences from the control group were slight and not statistically significant, with the following exceptions. Transient, significantly (p < 0.05 or p < 0.01) lower mean food consumption (g/kg/day values only) was noted in the 0.5 mg/kg bw/day group during Lactation Days 1–4 and in the 10 mg/kg/day group during Lactation Days 7–14, 17–21, and for the entire lactation treatment period (Lactation Days 1–21) compared to the control group. However, g/animal/day food consumption values in these groups were similar to the control group and mean body weights and body weight gains in these groups were unaffected by test substance administration; therefore, the aforementioned differences were not considered test substance-related.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmic lesions indicative of toxicity were observed in any of the test substance-treated groups. All findings observed were typical in prevalence and appearance for laboratory mice of this age and strain.

Haematological findings:

no effects observed

Description (incidence and severity):

Main animals: There were no test substance-related effects on hematology and coagulation parameters. Differences from the control group were slight and not statistically significant.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Main animals: significantly (p < 0.05 or p < 0.01) higher ALP, ALAT, and triglyceride values were noted in the 50 mg/kg bw/day group F0 males compared to the control group. Significantly (p < 0.05 or p < 0.01) higher ALP and triglyceride values were also noted for the non-mated females in the 50 mg/kg bw/day group compared to the control group. These changes were associated with hepatocellular hypertrophy noted microscopically and were considered test substance-related and adverse. Significantly (p < 0.01) lower mean serum calcium levels were noted for mated F0 females in the 50 mg/kg bw/day group compared to the control group; while there was no corresponding change in thyroid/parathyroid weights, a relationship to test substance administration cannot be ruled out.
No other test substance-related effects on serum chemistry parameters were noted at any dosage level. Differences from the control group were slight and not statistically significant, with the following exception. A significantly (p < 0.05) lower mean ALAT value was noted for mated F0 females in the 10 mg/kg bw/day group compared to the control group; this difference did not occur in a dose-related manner, and therefore was not considered test substance-related. In addition, significantly (p < 0.01) lower total bilirubin was noted for the 10 and 50 mg/kg bw/day group F0 males compared to the control group. The values were within the reference ranges in the Charles River Ashland historical control data and in a direction of no known toxicological importance.
T4 analysis (main study males): test substance-related, significantly (p < 0.01) lower mean total T4 values were noted for F0 males in the 10 and 50 mg/kg bw/day groups compared to the control group; however, no corresponding changes in thyroid weights or microscopic findings in the thyroid were noted in F0 males at any dosage level, and therefore these changes were considered nonadverse. There were no test substance-related effects on thyroid hormone values in the F0 males at 0.5 mg/kg bw/day. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Main study animals: physiological obserrvations, home cage, handling, sensory, neuromuscular and open field parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Week 15 (males) or on Lactation Day 21 (females), with one exception: a significantly (p < 0.05) longer time to first step was noted in the 50 mg/kg bw/day group F0 females (0.6 seconds) compared to the control group (0.4 seconds); however, the difference from the control group was minimal and values for the males were similar to the control group, and therefore it was not considered test substance-related. In the 10 mg/kg bw/day group, a significantly (p < 0.05) higher mean grooming count was noted for males and a significantly (p < 0.05) lower mean rearing count was noted for females compared to the respective control groups; the aforementioned differences were noted in single sexes and did not occur in a dose-related manner, and therefore were not considered test substance-related.
Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test substance administration at all dosage levels when evaluated during Study Week 15 and Lactation Day 21. Values obtained from the 6 subintervals evaluated (0–10, 11–20, 21–30, 31–40, 41–50, and 51–60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the Charles River Ashland historical control data. Differences from the control group were slight, not statistically significant when analyzed by a repeated measures analysis, within the Charles River Ashland historical control data ranges, and/or did not occur in a dose-related manner, with the following exception. A significantly (p = 0.022) higher mean cumulative total count was noted for F0 females in the 50 mg/kg bw/day group compared to the control group. Because this difference occurred in a single sex and no changes in habituation were noted, it was not considered testsubstance-related. No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups when the F0 animals were evaluated during Study Week 15 or on Lactation Day 21.

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):

Main study animals: test substance-related microscopic findings were noted in the liver of the 0.5, 10, and 50 mg/kg bw/day group F0 males and females. Test substance-related changes included mild to moderate centrilobular hepatocellular hypertrophy (in 17 males and 17 females at the lowest dose level, compared to zero incidence in controls; severity from minimal to moderate), hepatocellular necrosis (minimal in one male and mild in one female at the lowest dose level), and/or brown pigment in the Kupffer cells and hepatocytes (at the highest dose level only). The hypertrophy was characterized as increased hepatocellular size that was predominantly located in the centrilobular region of the hepatic lobule but extended to the periportal regions in the most severely affected sections. Single cell to coalescing hepatocyte necrosis was also observed in all dose groups. In the 50 mg/kg bw/day group F0 males and females only, 19 of 20 males and 5 of 19 females, respectively, minimal pigment accumulation was observed in the Kupffer cells and in the hepatocytes. The aforementioned findings correlated with higher alkaline phosphatase (ALP) and alanine aminotransferase (ALAT) in the 50 mg/kg bw/day group males and higher ALP in the non-mated females. Therefore, the constellation of changes in the liver was considered adverse in the F0 generation at all dose groups. There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The mean lengths of estrous cycles in the treatment groups were similar to the control group value.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

No test substance-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance-treated groups. Two, 0, 1, and 3 males in the control, 0.5, 10, and 50 mg/kg bw/day groups, respectively, did not sire a litter. Two, 0, 1, and 3 females in these same respective groups were determined to be nongravid. Although the mean values for the female fertility, male copulation, and female conception indices were outside of the Charles River Ashland historical control data ranges, these values were not statistically significant and there were no test substance-related changes in organ weights or microscopic examination of any reproductive organs. The mean numbers of days between pairing and coitus and gestation length in the test substance-treated groups were similar to the control group value. There were no test substance-related effects noted on the mean numbers of implantation sites and unaccounted-for sites in F0 females at any dosage level.

Effect levels (P0)

1

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects on reproduction at the highest tested dose

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Pups (during weaning): the general physical condition (defined as the occurrence and severity of clinical findings) of F1 pups in the 0.5 and 10 mg/kg bw/day groups was unaffected by test substance administration. Test substance-related increased incidences of digits missing from the left and/or right limbs, malrotation of the forelimbs, and small stature were noted in the 50 mg/kg bw/day group compared
to the control group. The increased number of pups that were found dead in the 50 mg/kg/day group was considered test substance-related.
F1 generation post-weaning: no test substance-related clinical observations were noted at any dosage level. Clinical observations noted in the test substance-treated groups at the daily examinations, detailed physical examinations, and approximately 1 hour following dose administration, including pale extremities, partial closure and/or enophthalmus of the eyes, and hair loss and/or scabbing on various body surfaces, were noted infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Pups (during weaning):
Test substance-related and adverse lower postnatal survival was noted for the 50 mg/kg bw/day group compared to the control group during PND 1–4 (pre-selection) and PND 4 (post-selection)–7 and resulted in lower postnatal survival from birth to PND 4 (pre-selection) and PND 4 (post-selection) to PND 21; differences from the control group were not statistically significant. One female in the 50 mg/kg bw/day group had a total litter loss on PND 5, while postnatal survival was less than 90% for 3 and 6 other females in this group during PND 1-4 and 4-7, respectively. All females in the control group had 100% postnatal survival during these intervals. One (1), 8(3), 3(2), and 28(10) pups (litters) in the control, 0.5, 10, and 50 mg/kg bw/day groups, respectively, were found dead. One (1), 5(4), 0(0), and 2(1) pups (litters) in the same respective groups were missing and presumed to have been cannibalized. The increased number of pups that were found dead in the 50 mg/kg/day group was considered test substance-related.
F1 generation post-weaning: one male in the 50 mg/kg bw/day group was found dead on PND 22; no significant clinical or macroscopic observations were noted for this male. In the 10 mg/kg bw/day group, 1 F1 male and 1 F1 female were euthanized in extremis on PND 21 following clinical observations of small stature, pale and cool body, and cool extremities on the day of euthanasia; no macroscopic findings were noted for these animals at necropsy. Limited tissues were examined for the aforementioned animals, and the cause of death/moribundity could not be determined. The aforementioned death and moribundity were considered test substance-related and secondary to their ability to thrive following weaning due to small stature and delayed development. In the 50 mg/kg/day group, one male was found dead on PND 28; at necropsy, this male was noted with dark red areas on the lungs, lungs not fully collapsed, and foamy contents in the trachea. Based on the macroscopic findings for this male, this death was attributed to a possible gavage error and not considered test substance-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Pups (during weaning): mean male and female pup birth weights (PND 1) in the 50 mg/kg bw/day group were lower (7.2% and 3.8%, respectively) than the control group; the difference was significant (p < 0.05) for males. Lower (generally significant [p < 0.01]) mean male and female F1 pup body weight gains were noted in the 50 mg/kg bw/day group during PND 1–7 and 17–21 compared to the control group; mean pup body weight gains in this group were similar to the control group during PND 7–17. Due to the lower mean birth weights and lower body weight gains, mean male and female F1 pup body weights in the 50 mg/kg bw/day group were lower (up to 23.2% and 21.6%, respectively) than the control group during PND 4-21; differences were generally significant (p < 0.01). The aforementioned offspring body weight effects noted in the 50 mg/kg bw/day group were considered test substance-related and adverse.
Mean male and female F1 pup body weights and body weight changes in the 0.5 and 10 mg/kg bw/day groups were unaffected by parental administration of the test substance. No statistically significant differences from the control group were noted.
F1 generation post-weaning: Mean body weights in the 50 mg/kg/day group F1 males and females were lower (11.9% and 17.2%, respectively) than the control group on PND 22 due to the body weight decrements noted in this group prior to weaning; the difference was significant (p < 0.01) for females. Test substance-related, significantly (p < 0.01) lower mean body weight gains were noted for F1 males and females in the 50 mg/kg bw/day group during PND 22–28; mean body weight gains for males and females in this group were similar to the control group for the remainder of the post-weaning treatment period and when the entire treatment period (PND 22–43) was evaluated. As a result of the initially lower mean body weights and due to the decrements in mean body weight gain
during the first week following weaning, mean body weights for the 50 mg/kg bw/day group F1 males and females were lower (up to 15.9% and 18.0%, respectively) during PND 28–43; differences were significant (p < 0.05 or p < 0.01) during PND 28–35 for males and PND 28–43 for females.
Mean body weights and body weight gains for F1 males and females in the 0.5 and 10 mg/kg bw/day groups were unaffected by test substance administration. Differences from the control group were slight and not statistically significant, with the following exception. A significantly (p < 0.05) lower mean body weight was noted in the 10 mg/kg bw/day group F1 females on PND 43 compared to the control group; this transient difference was not considered test substance-related.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

F1 generation post-weaning: evaluation of food consumption in the 0.5, 10, and 50 mg/kg bw/day groups was precluded due to numerous food spills; however, there generally did not appear to be any test substance-related effects on food consumption based on the limited data available.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis: there were no test substance-related effects on thyroid hormone values in the F1 males and females at any dosage level on PND 21. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.
No other clinical chemistry evaluations were performed.

Urinalysis findings:

not examined

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

Balanopreputial separation: mean ages of attainment of balanopreputial separation and mean body weights at the age of attainment were unaffected by F0 parental test substance administration. The mean ages of attainment of balanopreputial separation were 30.2, 29.5, and 31.0 days in the 0.5, 10, and 50 mg/kg bw/day groups, respectively, compared to 30.2 days in the control group; none of the differences from the control group were statistically significant. Mean body weight at the age of attainment was significantly (p < 0.05) lower in the 50 mg/kg bw/day group compared to the control group; this difference corresponded to the test substance-related, lower mean weekly body weights noted for males in this group, but had no impact on the age of attainment of balanopreputial separation. Mean body weights on the day of attainment in the 0.5 and 10 mg/kg bw/day groups were similar to the control group.
Vaginal patency: a significant (p < 0.05) delay in the mean age of vaginal patency was noted in the 50 mg/kg bw/day group (33.1 days) compared to the control group (29.9 days). This delay occurred in the presence of a lower (7.1%; not statistically significant) mean body weight on the day of attainment and mean body weights that were 10.5% to 18.0% lower than the control group throughout the post-weaning period (PND 22–43). Therefore, the delay in the mean age of attainment of vaginal patency in the 50 mg/kg bw/day group was considered secondary to the lower mean body weights noted in this group. Mean ages of attainment of vaginal patency and mean body weights at the age of attainment in the 0.5 and 10 mg/kg bw/day groups were unaffected by F0 parental test substance administration.
The mean ages of attainment of vaginal patency were 29.4 and 30.1 days in the 0.5 and 10 mg/kg bw/day groups, respectively, compared to 29.9 days in the control group. Mean body weights at the age of attainment were 18.7 g in both the 0.5 and 10 mg/kg bw/day groups compared to 19.7 g in the control group. None of the differences were statistically significant.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Pups at PND21: there were no test substance-related effects on thyroid weights in the F1 males and females at any dosage level on PND 21. Differences from the control group were considered to be the
result of normal biological variation and were not considered to be of toxicological significance.
F1 generation post-weaning (sacrifice on day 42): test substance-related effects on organ weights were observed in the 10 and 50 mg/kg bw/day group F1 males and 50 mg/kg bw/day group F1 females. In addition, test substance-related, significantly (p < 0.01) lower final body weight was observed in the 50 mg/kg bw/day group F1 females. Test substance-related higher liver weights (absolute and relative to brain and body weight) were observed in the 10 and 50 mg/kg bw/day group F1 males and the 50 mg/kg bw/day group F1 females; differences were significant (p < 0.05 or p < 0.01). These changes correlated to the microscopically observed hepatocellular hypertrophy seen in these groups and were considered adverse.
Test substance-related lower kidney weights (absolute and relative to brain and body weight) were observed in the 50 mg/kg bw/day group F1 males and females; differences were generally significant (p < 0.05 or p < 0.01). Test substance-related, significantly (p < 0.01) lower adrenal gland weights and spleen weights (absolute and relative to brain and body weight) were observed in the 50 mg/kg/day group F1 females. The aforementioned findings lacked a histologic correlate and were not considered adverse. There were no other test substance-related effects on organ weights.
A significantly (p < 0.05) lower mean absolute brain weight in the 50 mg/kg bw/day group females was considered secondary to the lower final body weight in that group. Other differences from the control group were slight, not statistically significant, and/or were not observed in a dose-related manner.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

External observations: in general, a higher number of pups with digits missing from the left and/or right limbs, malrotation of the forelimbs, and small stature were noted for F1 pups in the 50 mg/kg bw/day group.
Internal observations:
Pups found dead: aside from the presence or absence of milk in the stomach, internal findings were limited to the following. The malformation of cleft palate (entire length) noted for 6(1) and 3(2) pups (litters)
in the 0.5 and 50 mg/kg bw/day groups, respectively. Because this finding did not occur in a dose-related manner, it was not considered test substance-related. In the 50 mg/kg bw/day group, one male pup was noted with 7th sternebrae and an accessory skull bone and one female was noted with severely malaligned sternebrae; these findings were noted in a single litter and therefore were not considered test substance-related.
Scheduled pup necropsy at PND21 (thyroid gland examination): no internal findings that could be attributed to parental test substance administration were noted at the necropsy of pups euthanized on PND 21. The only internal finding was an enlarged right parathyroid gland noted for one male in the 50 mg/kg/day group; because this finding was noted in a single animal, it was not considered test substance-related.
Pups at PND21 and pups euthanized due to death of dam: no internal findings that could be attributed to parental test substance administration were noted at the necropsy of nonselected pups or pups euthanized due to death of dam. The only internal finding was opacity of the left eye noted for one male in the 50 mg/kg bw/day group; this finding was not considered test substance-related because it occurred in a single animal.
F1 generation post-weaning: no test substance-related internal findings were observed at any dosage level for F1 males and females at the scheduled necropsy. Macroscopic findings observed in the test substance-treated groups occurred infrequently and/or in a manner that was not dose-related.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

PND21 females: the mammary glands were scored for each time point independently. Each gland was assigned a score of 1-4 with one being least developed and 4 being most developed. While the most
developed mammary glands were observed in the control group females, there were no discernable test substance-related differences between the 0 and 50 mg/kg bw/day group F1 females on PND 21.
F1 generation post-weaning (sacrifice on day 42): similar to the F0 generation, test substance-related microscopic findings were noted in the liver of the 10 and 50 mg/kg bw/day group males and females. Test substance-related changes included minimal to moderate centrilobular hepatocellular hypertrophy and hepatocellular necrosis and were considered adverse. In addition, minimal granulomatous inflammation was observed in some livers and was centered on foci of mineralization.
Mammary gland whole mounts were qualitatively evaluated on PND 43. The mammary glands were scored for each time point independently. Each gland was assigned a score of 1-4 with one being least developed and 4 being most developed. While the most developed glands were observed in the control group females at each time point, there were no discernable test substance-related differences between the 0 and 50 mg/kg bw/day group females at either time point.

Other effects:

no effects observed

Description (incidence and severity):

Anogenital distance (PND0): the anogenital distances (absolute and relative to the cube root of pup body weight) in the 0.5, 10, and 50 mg/kg bw/day groups were similar to the control group values. Differences from the control group were slight and not statistically significant.
Areola/nipple anlagen: areolae/nipple anlagen in the F1 male pups was unaffected by parental administration of the test substance when evaluated on PND 13. The test substance-treated group values were not
statistically significantly different from the control group values.
The mean number of pups born, live litter size, and the percentage of males at birth in the 0.5, 10, and 50 mg/kg bw/day groups were similar to the control group values.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

gross pathology

2

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Target system / organ toxicity (F1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Formulation analysis

The analyzed dosing formulations were within Charles River SOP range for solutions (90% to 110%), with the following exception. The results of the initial assessment of concentration acceptability of the 02 Nov 2016 low dose formulation failed to meet the acceptance criteria. Subsequent analysis of the back-up samples confirmed the results of the initial analysis and the overall mean concentration was reported as 117% of target. This had no impact on the study as this dosing formulation was only utilized for approximately 1 week of dose administration and the animals received a higher than anticipated dose (0.56 mg/kg) and there was still adequate separation of doses between 0.5 and 10 mg/kg bw/day.  The results of the analysis are presented in the table below.

Date of Preparation	Mean Concentration, mg/mL (% of Target)
	Group 1 (0 mg/mL)	Group 2 (0.1 mg/mL)	Group 3 (2 mg/mL)	Group 4 (10 mg/mL)
19 Oct 2016	NQ (NA)	0.105 (105)	2.10 (105)	9.75 (97.5)
02 Nov 2016	NQ (NA)*	0.117 (117)	1.94 (97.0)	9.63 (96.3)
23 Nov 2016	NQ (NA)	0.101 (101)	2.15 (107)	11.0 (110)
26 Feb 2017	NQ (NA)	0.0995 (99.5)	1.98 (99.2)	10.5 (105)
NQ = Not quantifiable NA = Not applicable. * = Analyzed in a run which was not valid.

Applicant's summary and conclusion

Conclusions:

In a combined 90-day toxicity study with reproductive/developmental toxicity screening with mice, conducted according to GLP and OECD guidelines 408 and 422, reproductive performance was not affected up to and including the highest dose level of 50 mg/kg bw/day. Therefore the NOAEL for reproductive toxicity was considered to correspond to 50 mg/kg bw/day. At this dose level, the lower postnatal survival in combination with lower mean pup birth weights and weight gain, as well as a higher number of pups with digits missing from the left and/or right limbs, malrotation of the forelimbs, and small stature were noted. Based on this the NOAEL for developmental toxicity was set at 10 mg/kg bw/day. Adverse liver findings (hepatocellular hypertrophy in combination with hepatocellular necrosis) in combination with higher liver weights (absolute and relative) were noted in F1 animals treated until PND 42. Based on this the NOAEL for systemic toxicity in F1 generation was set at 0.5 mg/kg bw/day.