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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

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:

other: OECD Guideline 422 (Combine 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 (incl. QA statement)

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 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 the 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

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.

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

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.

Duration of test:

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).

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.

Examinations

Maternal 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 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.

FOOD CONSUMPTION: yes
- Time schedule for examinations: main study females: 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.

- 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 (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 (lactation day 21 for mated females).
- 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.
- 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 (lactation day 21 for mated females).
- 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.
- 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: 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).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on lactation day 21 (euthanasia on day 130-142); for females that failed to deliver: following 90-day treatment, mating and until postmating day 23 (euthanasia on day 113)

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 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.

OTHER:
Oestrous cycle: 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.

Ovaries and uterine content:

The ovaries and uterus were examined after termination: Yes
Examinations included:
- Gravid uterus weight: no, females were allowed to litter naturally
- Number of corpora lutea: no
- Number of implantations: yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

No fetal examinations were performed, females were allowed to litter naturally. Gross necropsy was performed on all pups not selected for F1 generation on PND 21 and all F1 generation animals. Furthermore, gross necropsy was conducted 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.

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.

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.

Historical control data:

Testing laboratory historical control data on developmental and reproductive toxicity in the same animal strain are available from a period of 1997 - 2015.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

Main study animals: no test substance-related clinical observations were noted for F0 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):

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.
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):

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):

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 bw/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 bw/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):

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):

Significantly (p < 0.05 or p < 0.01) higher ALP and triglyceride values were 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.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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 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 therefore it was not considered test substance-related. In the 10 mg/kg bw/day group, a significantly (p < 0.05) lower mean rearing count was noted for females compared to the respective control groups; the aforementioned difference was 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):

Test substance-related effects on organ weights were observed in the 10 and 50 mg/kg bw/day group F0 females. Significantly (p < 0.01) higher liver weights (absolute and relative to body and brain weight) were observed in 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):

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 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.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related microscopic findings were noted in the liver of the 0.5, 10, and 50 mg/kg bw/day group F0 females. Test substance-related changes included mild to moderate centrilobular hepatocellular hypertrophy (in 17 females at the lowest dose level, compared to zero incidence in controls; severity from minimal to moderate), hepatocellular necrosis (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 females, in 5 of 19 females 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 on estrous cycle duration were observed in the test groups in comparison with control groups. For additional details on general and reproductive toxicity see sections 7.5.1 and 7.8.1 of this IUCLID (cross-references).

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No statistically significant differences between the treated and control groups were noted. Two, 0, 1, and 3 females in controls, 0.5, 10 and 50 mg/kg bw/day 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 unaccounted-for sites and implantation sites in the 0.5, 10, and 50 mg/kg bw/day groups were similar to the control group values. The number of unaccounted-for sites was calculated for each female that delivered by subtracting the number of pups born from the number of former implantation sites observed.

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Description (incidence and severity):

No difference between the number of live born pups was observed between the treatment and control groups.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Mean gestation lengths in the 0.5, 10, and 50 mg/kg/day groups were similar to those in the control group.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Mean gestation lengths in the 0.5, 10, and 50 mg/kg/day groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No statistically significant differences between the treated and control groups were noted. Two, 0, 1, and 3 females in controls, 0.5, 10 and 50 mg/kg bw/day 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.

Effect levels (maternal animals)

Key result

Dose descriptor:

LOAEL

Remarks:

maternal toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Maternal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: liver

Description (incidence and severity):

Test substance-related changes included mild to moderate centrilobular hepatocellular hypertrophy (in 17 females at the lowest dose level, compared to zero incidence in controls; severity from minimal to moderate) and hepatocellular necrosis (mild in one female at the lowest dose level).

Results (fetuses)

Fetal body weight changes:

not examined

Description (incidence and severity):

No fetal body weights were examined, females were allowed to litter naturally.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): 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.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The mean number of pups born and live litter size were similar to the control group values.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The percentage of males at birth was similar between the treated and control groups.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

The mean numbers of live litter size were similar to the control group values. Offspring pup birth weights were affected at 50 mg/kg bw/day (7.2% and 3.8% lower in males and females, respectively, in comparison to the controls; statistically significant for males).

Changes in postnatal survival:

effects observed, treatment-related

Description (incidence and severity):

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.
Postnatal survival in the 0.5 and 10 mg/kg bw/day groups was unaffected by test substance administration.

External malformations:

no effects observed

Description (incidence and severity):

No external malformations were noted; however, 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.

Skeletal malformations:

not examined

Description (incidence and severity):

Not specifically examined, but no malformations were noted at gross necropsy.

Visceral malformations:

not examined

Description (incidence and severity):

Not specifically examined, but no malformations were noted at gross necropsy.

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.
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.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

changes in postnatal survival

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

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 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 test with reproductive and developmental toxicity screening, conducted according to GLP and OECD guidelines 408 and 422, at the highest dose level of 50 mg/kg bw/day lower pup birth weights, subsequent lower weight gains and reduced postnatal survival were observed. Also 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. In parental animals, 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. Based on this the NOAEL for developmental toxicity was set at 10 mg/kg bw/day, while the lowest dose level of 0.5 mg/kg bw/day was considered to be the LOAEL for maternal toxicity.