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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

21 Sep 1988

Deviations:

no

Test guideline 2

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

Details on route of administration:

The selected route of administration for this study was oral (gavage) because this a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.

Vehicle:

water

Details on oral 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

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 doses 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.

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

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.
- Rationale for selecting satellite groups: Five females in the control and high-dose groups were not mated, but were administered the test substance throughout the study and were utilized for gender comparison without gestation influence. 15 animals/sex/dose were assigned to the clinical pahology group, in order to obtain terminal collections for hematology, serum chemistry, and coagulation parameters (5/sex/group).
- Post-exposure recovery period in satellite groups: none
- Section schedule rationale: males following 90-day treatment and the mating period (euthanasia 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; pups: 21 days post-weaning (euthanasia on day 42).

Positive control:

Not applicable.

Examinations

Observations and examinations performed and frequency:

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

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)

URINALYSIS: No

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).

IMMUNOLOGY: No

OTHER:
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.

Thyroid hormone analysis (F0 only, for results on F1 generation see sections 7.8.1 and 7.8.2 of this IUCLID (cross-references)): 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.

For details on reproductive and developmental toxicity examinations and pups examinations see sections 7.8.1 and 7.8.2 of this IUCLID (cross-references).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, 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.
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.

HISTOPATHOLOGY: Yes, 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, lymph 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.

Other examinations:

For reproductive and developmental toxicity examinations see sections 7.8.1 and 7.8.2 of this IUCLID (cross-references).

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.

Results and discussion

Results of examinations

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.
Clinical pathology animals:
One female in the 10 mg/kg bw/day group was removed from study on Study Day 1 due to a clinical finding of swollen urogenital area following dosing on Study Day 0; at necropsy, this female was noted with dark red discoloration of the lungs, clear fluid contents in the uterus, and an enlarged vagina with thick white contents. This euthanasia was unrelated to the test substance. An additional female was subsequently added to the 10 mg/kg bw/day group of the clinical pathology phase.
In the control group, 1 female was euthanized in extremis on Study Day 61 following persistent scabbing on the neck and a body weight loss of 5.9% during Study Days 49–56; remarkable clinical observations for this female included scabbing and hair loss on the dorsal head and/or neck during Study Days 13–61. At necropsy, this female was noted with scabbing on the dorsal head, mottled and rough surfaces on the lungs, and cystic ovaries. All males and all other females in the clinical pathology phase survived to the scheduled euthanasia.

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.
Clinical pathology animals: mean body weights and body weight gains in the 0.5, 10, and 50 mg/kg bw/day group males and females in the clinical pathology phase were unaffected by test substance administration throughout the study.

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/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.
Clinical pathology animals: mean food consumption, evaluated as g/animal/day and g/kg/day, in the 0.5, 10, and 50 mg/kg bw/day group males and females in the clinical pathology phase was unaffected by test substance administration throughout the study.

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 pahtology 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.
Clinical pathology animals: Test substance-related effects on serum chemistry parameters included higher mean ASAT values noted for males in the 50 mg/kg bw/day group and females at all dosage levels, higher mean ALAT values in the 10 and 50 mg/kg bw/day group males and females, and higher mean ALP values in the 50 mg/kg bw/day group males and females compared to the control group; differences were not statistically significant. The aforementioned differences correlated to the higher liver weights and adverse liver findings of hepatocellular hypertrophy with hepatocellular necrosis noted in the main study F0 males and females at 10 and 50 mg/kg bw/day. There were no test substance-related effects on serum chemistry parameters at 0.5 mg/kg bw/day. Differences from the control group were slight and not statistically significant.

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

Description (incidence and severity):

Main study animals: test substance-related effects on organ weights were observed in the 10 and 50 mg/kg bw/day group F0 males and females. Significantly (p < 0.01) higher liver weights (absolute and relative to body and brain weight) were observed in the 10 and 50 mg/kg bw/day group F0 males, the 50 mg/kg bw/day group non-mated F0 females, and the 10 and 50 mg/kg bw/day group F0 females necropsied on Lactation Day 21. The higher liver weights noted in these groups correlated to the microscopically observed hepatocellular hypertrophy and were considered adverse.
There were no other test substance-related effects on organ weights. Other differences from the control group were slight, not statistically significant, and/or did not occur in a dose-related manner.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Main study animals: no test substance-related internal findings were observed at any dosage level in females that failed to deliver, the female with total litter loss or males and females (mated and non-mated) 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.
Clinical pathology animals: no test substance-related internal findings were observed at any dosage level in clinical pathology phase 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.

Neuropathological findings:

not examined

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

Description (incidence and severity):

No effects were observed on the mean length of estrous cycle. For details on reproductive and developmental effects see sections 7.8.1 and 7.8.2 of this IUCLID (cross-references).

Effect levels

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

Target system / organ toxicity

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

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 formulation 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 GLP compliant 90-day study with reproductive and developmental toxicity screening with mice, performed according to OECD guidelines 408 and 422, adverse effects in the liver (hepatocellular hypertrophy with hepatocellular necrosis) were observed starting from the lowest dose level of 0.5 mg/kg bw/day. Associated changes in clinical chemistry included higher ALP, ALAT and triglyceride values in high-dose males and higher ALP and triglyceride values in high-dose non-mated females, and higher relative liver weights at 10 and 50 mg/kg bw/day in both sexes. These changes were considered to be adverse. Therefore the lowest dose level of 0.5 mg/kg bw/day was considered to be the LOAEL for systemic toxicity in parental generation.