Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 September 2016 - 12 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 September 2016 - 12 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
October 2008
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Version / remarks:
July 2000
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3050 (Repeated dose 28-day oral toxicity study in rodents)
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Stability in corn oil for at least 6 hours at room temperature is confirmed over the concentration range 1 to 200 mg/mL Stability in corn oil for at least 8 days in the refrigerator is confirmed for the concentration 200 mg/mL.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was heated up to a maximum temperature of 69 ºC for a maximum of 2 hours and 27 minutes before formulating.
Species:
rat
Strain:
other: Crl:WI(Han)
Remarks:
outbred, SPF-quality
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males ca. 10 weeks, females ca. 13 weeks (at the start of F0 treatment)
- Weight at study initiation: males 277-302 g, females 201-239 g
- Fasting period before study: none
- Housing: sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied.
At pretest: females were housed in groups of 5/cage in Macrolon type IV plastic cages
Pre-mating: in groups of 5/sex/dose in Macrolon type IV plastic cages
Mating: on 1:1 basis m:f in Macrolon type III plastic cages
Post-mating: males in their home cages (5/dose), females individually in Macrolon type III plastic cages
Lactation: females were housed with pups in Macrolon type III cages. During locomotor activity measurements pups were kep warm in their home cages using warm water bottles. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
Locomotor activity monitoring: individually in a Hi-temp cages without cage enrichment, bedding material, food and water
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum, except during motor activity measurements for max. 2 hours and overnight before terminal sacrifice
- Water: tap water, ad libitum, except during motor activity measurements for max. 2 hours
- Acclimation period: at least 5 days

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

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 12 September 2016 To: 12 January 2017
Route of administration:
oral: gavage
Details on route of administration:
This study should provide part of a rational basis for toxicological risk assessment in man. The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
corn oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: The test item was heated up to a maximum temperature of 69 ºC for a maximum of 2 hours and 27 minutes before formulating. High-dose formulations (w/w) were heated to 69 °C for a maximum duration of 5 hours and 40 minutes under continuous stirring, until a visually homogenous formulation was obtained. After cooling down, these formulations were stored in the refrigerator for at least an overnight period, but maximally 8 days. Low- and mid-dose formulations (w/w) were prepared by diluting the high-dose formulation with the required amount of vehicle daily within 6 hours prior to dosing, and were homogenized to a visually acceptable level. High-dose formulation was acclimated to room temperature conditions and made visually homogenous prior to this dilution.
Adjustment was made for specific gravity of the vehicle. Density of the high-dose formulation was determined on a single occasion during the treatment period (0.93 g/mL), after the formulation had been acclimatized to room temperature conditions on the day of preparation of the low- and mid-dose concentrations. Until then a density value of 0.92 g/mL was taken for the high-dose formulation (i.e. the same density as for the vehicle). No correction was made for the purity/composition of the test item.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations performed at Charles River Den Bosch
- Amount of vehicle (if gavage): 5 mL/kg bw
- Adjustment was made for specific gravity of the vehicle (0.92 g/mL).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during pretreatment phase (15 November 2016) according to a validated method (UPLC-MS). Samples were collected and analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Duration of treatment / exposure:
Males: 29 days (2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy)
Females that delivered: 50-56 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy
Females which failed to deliver healthy offspring: 42-53 days
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle controls (Group 1)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results of a 10-day dose range finding study conducted at the same test facility
- Rationale for selecting satellite groups: 5 animals/sex/group were selected for functional observations, locomotor activitiy, clinical pathology, macroscopic exmination, organ weights and histopathology
- Section schedule rationale (if not random):
Males: following the completion of the mating period (a minimum of 28 days test substance administration)
Females which delivered: PND 14-16
Females that failed to deliver: Post-coitum Days 25-28 (females with evidence of mating) or approximately 25 days after the last day of the mating period (female without evidence of mating).
Females with total litter loss: within 24 hours of litter loss
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for mortality and viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

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

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

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to the scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters examined: WBC, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), RBC, reticulocytes, RDW, haemoglobin, haematocrit, MCV, MCY, MCHC, platelets; prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled necropsy
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters: ALAT, ASAT, ALP, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: the selected males during week 4 of treatment; the selected females during the last week of lactation (PND 6-13).
- Dose groups that were examined: all, on selected 5 animals/sex/dose
- Battery of functions tested: sensory activity (hearing ability, pupillary reflex, static righting reflex), grip strength (fore- and hind-limbs), motor activity (total movement and ambulations)

IMMUNOLOGY: No

OTHER:
Oestrous cycle examinations: Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period.

Thyroid hormone measurements:
Serum T4 concentration was measured in males prior to scheduled sacrifice

For details on the examinations on pups see section 7.8.1 and 7.8.2 of this IUCLID (cross-references).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, full post-mortem necropsy, with special attention paid to the reproductive organs. The following organ weights were recorded:
- On selected 5 animals/sex/group: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostates, seminal vesicles including coagulating glands, spleen, testes, thymus, thyroid including parathyroid if detectable, uterus including cervix.
- All remaining animals: epididymides, prostate, seminal vesicles including coagulation glands, testes, thyroid

HISTOPATHOLOGY: Yes
- On selected 5 animals/sex/group in control and high-dose groups and all animals that were euthanized in extremis: adrenal glands, brain (cerebellum, mid-brain, cortex), caecum, cervix, clitoral gland, colon, coagulation gland, duodenum, epididymides, eyes (with optic nerve if detectable and Harderian gland), mammary gland area (males and females), femur including joint, heart, ileum, jejunum, kidneys, liver, lung, lymph nodes (mandibular, mesenteric), ovaries, Peyers' patches if detectable, pituitary gland, preputial gland, prostate gland, rectum, sciatic nerve, seminal vesicles, skeletal muscles, spinal cord (cervica, midthoracic, lumbar), spleen, sternum with bone marrox, stomach, testes, thymus, thyroid including parathyroid if detectable, trachea, urinary bladder, uterus, vagina, all gross lesions.
- On selected 5 males in all groups and all males that failed to sire: additional testes slides
- On all animals: all gross lesions
- All selected males of low- and mid-dose groups: thymus, spleen and sternal bone marrow, based on possible effects on these organs in the high-dose group
- All selected males and females of low- and mid-dose groups: liver, urinary bladder, based on possible effects on these organs in the high-dose group

For details on the examinations on pups see section 7.8.1 and 7.8.2 of this IUCLID (cross-references).
Other examinations:
For details on the examinations on pups see section 7.8.1 and 7.8.2 of this IUCLID (cross-references).
Statistics:
The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data
• The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period.
Salivation seen in a dose-related incidence after dosing among animals of the control and treated groups was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). For the test item treated groups, this sign may be related to taste of the test item.
All other clinical signs among surviving animals occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were not considered to be signs of toxicological relevance. No findings were noted during the arena observations in this study.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test item.
One control male was sacrificed in extremis on Day 28 due to a serious eye injury. At clinical observation, the left eye appeared exophthalmic and dehydrated and was discolored black. Additionally, the right cheek was thickened. At necropsy, the left eye was recorded as desiccated, with marked acute necrotizing inflammation as microscopic correlate.
One female at 750 mg/kg bw/day was sacrificed on PND 1 due to total litter loss.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day, males had a slightly lower weight gain throughout the treatment period, achieving a level of statistical significance on Day 8 of the premating period only. Mean body weight at the end of treatment were approximately 5% lower than the control group.
Body weights and body weight gain of other treated groups remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was not considered affected by treatment.
The statistically significant higher relative food intake of females at 750 mg/kg bw/day over Days 11-14 of the post-coitum period was considered unrelated to treatment, since this change was slight and not consistently present with continuing treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day, the following (statistically significant) changes in haematology parameters distinguished treated animals from control animals:
- Lower relative lymphocyte counts in males (not statistically significant).
- Higher relative monocyte counts in males.
- Higher relative eosinophil counts in males.
- Lower red blood cell counts in males.
- Higher reticulocyte counts in males (not statistically significant), primarily attributed to a high value of one male
- Lower haemoglobin in males.
- Lower haematocrit in males.
- Lower mean corpuscular volume (MCV) in females.
- Lower mean corpuscular haemoglobin (MCH) in females.
The statistically non-significant lower activated partial thromboplastin time of males at 750 mg/kg bw/day was not considered to be of toxicological relevance since the opposite effect (i.e. an increase) would be expected in case of target organ toxicity.
The statistically significant lower red cell distribution width of females at 250 mg/kg bw/day was not considered to be related to treatment as this occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher alanine aminotransferase activity (ALAT) in males at 250 mg/kg bw/day (not statistically significant) and in males and females at 750 mg/kg bw/day (not statistically significant for females; higher mean primarily due to one animal).
- Higher aspartate aminotransferase activity (ASAT) in males at 250 mg/kg bw/day (not statistically significant; primarily due to one animal) and in males and females at 750 mg/kg bw/day (not statistically significant for females; higher mean primarily due to one animal).
- Higher alkaline phosphatase activity (ALP) in males at 750 mg/kg bw/day
- Higher total protein in males at 750 mg/kg bw/day
- Higher total bilirubin in males at 750 mg/kg bw/day (not statistically significant; higher mean primarily due to one male) and in one female at 750 mg/kg bw/day (not statistically significant)
- Higher urea in males at 750 mg/kg bw/day.
- Higher creatinine in males at 750 mg/kg bw/day.
- Higher cholesterol in males at 750 mg/kg bw/day.
- Higher bile acids in three males and in one female at 750 mg/kg bw/day (not statistically significant for both sexes).
- Higher sodium in females at 750 mg/kg bw/day.
- Lower chloride in males at 750 mg/kg bw/day
Any other statistically significant changes in clinical biochemistry parameters were not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.
Serum levels of T4 in F0 males were not considered to be affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day, females had a statistically significantly lower motor activity for both total movements and ambulations. At 250 mg/kg bw/day, motor activity (total movements only) was statistically significantly higher than controls.
All groups showed a similar habituation profile in motor activity, with high activity in the first interval with a decreasing trend in activity over the duration of the test period. Other functional observation parameters were not considered to be affected by treatment. Hearing ability, pupillary reflex and static righting reflex were normal in all selected animals. Fore- and hindlimb grip strength was similar between control and treated groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant test item-related higher organ weights were observed at 750 mg/kg bw/day in liver of both sexes and spleen of males. Statistically significant test item-related lower thymus weights (males) were noted at 750 mg/kg bw/day. Test item-related higher liver weights (absolute and relative to body weights) were noted in the 750 mg/kg bw/day group males and females (relative to body weight in males 35% higher than in controls, in females 21% higher than in controls, p < 0.01). Test item-related higher spleen weights (absolute and relative to body weights) were noted in the 750 mg/kg bw/day group males (relative to body weight 34% higher than in controls, p < 0.01). Test item-related lower thymus weights (absolute) were noted in the 750 mg/kg bw/day group males (30% lower than in controls, not statistically significant for relative to body weight).
Any other differences, including those that reached statistical significance were considered not to be treatment-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
A test item-related macroscopic finding was noted in the liver of a 750 mg/kg bw/day male, consisting of many reddish foci of the right and left median lobes.
Findings of note were recorded for one 250 mg/kg bw/day male and consisted of several yellowish foci or soft nodules in the lung, pleura of the thoracic cavity and thymus, and a thickened thymus. The microscopic correlates were inflammatory processes of lung pleura and thymus capsule, and were considered to be gavage-related.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with the test substance were noted starting at 250 mg/kg bw/day in the liver of both sexes and spleen of males, and at 750 mg/kg bw/day in the urinary bladder of both sexes and thymus of males.
In the liver, bile duct hypertrophy/hyperplasia was observed starting at 250 mg/kg bw/day in males (up to moderate) and females (up to marked). Peribiliary inflammatory cell infiltrate was observed starting at 250 mg/kg bw/day in males (up to marked) and females (up to slight). Coagulative necrosis was observed starting at 250 mg/kg bw/day in one male (minimal) and in 4 males at 750 mg/kg bw/da (up to moderate) and at 750 mg/kg bw/day in 2 females (up to slight). Peribiliary fibrosis was observed at 750 mg/kg bw/day in all 5 males (up to moderate) and all 5 females (up to slight).
In the urinary bladder, urothelium hyperplasia was observed at increased incidence and severity at 750 mg/kg in all males (up to slight) and all females (up to slight). The hyperplasia was in general diffuse and without cellular atypia.
In thymus, lymphoid atrophy and increased lymphocytosis were observed in 750 mg/kg bw/day males (minimal).
In the spleen, hematopoiesis was observed at increased incidence and severity in all males starting at 250 mg/kg bw/day (up to moderate).
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Estrous cycle:
At 750 mg/kg bw/day, two females at 750 mg/kg bw/day were acyclic; one of these females did not mate and another one had a normal litter. Acyclic estrous cycles are infrequent occurrences in this type of study (1/316 historical control dams, period 2015-2017).
Length and regularity of the estrous cycle at 75 and 250 mg/kg bw/day were not considered to have been affected by treatment. Most females had regular cycles of 4 days. Extended di-estrus occurred in two females at 750 mg/kg bw/day with a regular cycle. An irregular cycle was also noted for one female at 750 mg/kg bw/day with normal litter. Given their incidental nature and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment. However, in combination with elongated precoital time in two other females at 750 mg/kg bw/day and developmental effects these findings may indicate adverse effect on reproduction (see cross-references).
Details on results:
For details on the reproductive and developmental toxicity see sections 7.8.1 and 7.8.2 of this IUCLID (cross-references).
The effects in the thymus and spleen were considered non-adverse by the study director. Lower thymus weight, atrophy and increased lymphocytolysis in males at 750 mg/kg bw/day were considered non-adverse based on the observed minimal severities. Increased incidence and severity of hematopoiesis in the spleen of 250 and 750 mg/kg bw/day males, correlated with higher spleen weights and haematological changes including lower blood cell counts, haemoglobin and haematocrit and higher reticulocyte counts at 750 mg/kg bw/day in males, were considered non-adverse based on the absence of inflammatory, degenerative or proliferative lesions. However, the latter effects are considered to be relevant for risk assessment purposes. Nevertheless, considering these effects does not change the setting of the NOAEL.
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
bile duct
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Analytical determination of the test formulations:

Mean accuracies in the low-, mid- and high-dose groups were 100, 11 and 108%, respectively (n = 6, 2, 6). For the mid-dose formulation prepared prior to treatment, the mean accuracy was above the

target concentration (i.e. 111% of target). Since this value only marginally exceeded the range of 90-110% of nominal, it was considered acceptable for the purpose of this study.

The coefficient of variation for low- and high-dose groups was 16 and 3.0%, respectively. ≤ 10%). The criterion for homogeneity is that the coefficient of variation ≤10%. For the low-dose formulation prepared prior to treatment, the coefficient of variation was above the target concentration (i.e. 16% of target). This was mainly due to two lower accuracy values (79 and 80%) at 90% sampling height. Overall, these results were considered acceptable for the purpose of this study.

Conclusions:
In the GLP-compliant OECD guideline 422 study with rats that received the test substance in corn oil at dose levels of 0, 75, 250 and 750 mg/kg bw/day, adverse effects on the liver (hypertrophy/hyperplasia of the bile ducts, peribiliary inflammatory cell infiltrates and focal coagulative necrosis) were observed starting from the dose level of 250 mg/kg bw/day and increased in severity and incidence at 750 mg/kg bw/day. At 750 mg/kg bw/day they were accompanied by changes in clinical chemistry (higher ALAT and ASAT activities, higher bile acids and bilirubin levels, statistically significant in males) and liver weight changes. Based on these findings the NOAEL for general toxicity was set at 75 mg/kg bw/day.
Executive summary:

In the GLP-compliant OECD guideline 422 study, groups of 10 rats/sex/dose were treated with the test item in corn oil at dose levels of 0 (vehicle controls), 75, 250 and 750 mg/kg bw/day 2 weeks before mating, during mating and during gestation and lactation until PND 14-16 (females that delivered), for a minimum of 28 days (males) or 42 -56 days (females). Adverse effects on the liver (hypertrophy/hyperplasia of the bile ducts, peribiliary inflammatory cell infiltrates and focal coagulative necrosis (minimal in one male)) were observed starting from the dose level of 250 mg/kg bw/day and increased in severity and incidence at 750 mg/kg bw/day. At 750 mg/kg bw/day they were accompanied by changes in clinical chemistry (higher ALAT and ASAT activities, higher bile acids and bilirubin levels, statistically significant in males) and statistically significantly increased absolute and relative liver weights in both sexes. Coagulative necrosis was observed starting in 4 males at 750 mg/kg bw/day (up to moderate) and at 750 mg/kg bw/day in 2 females (up to slight). Furthermore, peribiliary fibrosis was observed at 750 mg/kg bw/day in all 5 males (up to moderate) and all 5 females (up to slight). Starting from 250 mg/kg bw/day increased incidence and severity of hematopoiesis in the spleen was observed in males, which correlated with higher spleen weights and haematological changes including lower blood cell counts, haemoglobin and haematocrit and higher reticulocyte counts at 750 mg/kg bw/day. However, these changes were considered non-adverse based on the absence of inflammatory, degenerative or proliferative lesions. Nevertheless, these effects are considered to be relevant for risk assessment purposes.

At 750 mg/kg bw/day urothelium hyperplasia was observed at increased incidence and severity in all selected males and females up to slight degree. Although the hyperplasia was in general diffuse and without cellular atypia, based on the proliferative character this finding was regarded as adverse.

Based on the observed effects, the NOAEL for general parental toxicity was set at 75 mg/kg bw/day.

Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 September 2016 - 12 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproductive/Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stability in corn oil for at least 6 hours at room temperature is confirmed over the concentration range 1 to 200 mg/mL Stability in corn oil for at least 8 days in the refrigerator is confirmed for the concentration 200 mg/mL.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was heated up to a maximum temperature of 69 ºC for a maximum of 2 hours and 27 minutes before formulating.
Species:
rat
Strain:
other: Crl:WI(Han)
Remarks:
SPF-quality, outbred
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: females ca. 13 weeks (at the start of F0 treatment)
- Weight at study initiation: females 201-239 g
- Fasting period before study: none
- Housing: sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied.
At pretest: females were housed in groups of 5/cage in Macrolon type IV plastic cages
Pre-mating: in groups of 5/sex/dose in Macrolon type IV plastic cages
Mating: on 1:1 basis m:f in Macrolon type III plastic cages
Post-mating: females individually in Macrolon type III plastic cages
Lactation: females were housed with pups in Macrolon type III cages. During locomotor activity measurements pups were kep warm in their home cages using warm water bottles. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
Locomotor activity monitoring: individually in a Hi-temp cages without cage enrichment, bedding material, food and water
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum, except during motor activity measurements for max. 2 hours and overnight before terminal sacrifice
- Water: tap water, ad libitum, except during motor activity measurements for max. 2 hours
- Acclimation period: at least 5 days

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

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item was heated up to a maximum temperature of 69 ºC for a maximum of 2 hours and 27 minutes before formulating. High-dose formulations (w/w) were heated to 69 °C for a maximum duration of 5 hours and 40 minutes under continuous stirring, until a visually homogenous formulation was obtained. After cooling down, these formulations were stored in the refrigerator for at least an overnight period, but maximally 8 days. Low- and mid-dose formulations (w/w) were prepared by diluting the high-dose formulation with the required amount of vehicle daily within 6 hours prior to dosing, and were homogenized to a visually acceptable level. High-dose formulation was acclimated to room temperature conditions and made visually homogenous prior to this dilution.
Adjustment was made for specific gravity of the vehicle. Density of the high-dose formulation was determined on a single occasion during the treatment period (0.93 g/mL), after the formulation had been acclimatized to room temperature conditions on the day of preparation of the low- and mid-dose concentrations. Until then a density value of 0.92 g/mL was taken for the high-dose formulation (i.e. the same density as for the vehicle). No correction was made for the purity/composition of the test item.

- VEHICLE
- Justification for use and choice of vehicle (if other than water):
based on trial formulations performed at Charles River Den Bosch
- Amount of vehicle (if gavage): 5 mL/kg bw
- Adjustment was made for specific gravity of the vehicle (0.92 g/mL).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during pretreatment phase (15 November 2016) according to a validated method (UPLC-MS). Samples were collected and analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: a maximum of 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually in Macrolon type III plastic cage with sawdust as bedding material
Duration of treatment / exposure:
Females that delivered: 50-56 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy
Females which failed to deliver healthy offspring: 42-53 days
Pups were not treated, but could have been exposed to the test substance via lactation.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose
Duration of test:
Females that delivered: 50-56 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy
Females which failed to deliver healthy offspring: 42-53 days
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle controls (Group 1)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results of a 10-day dose range finding study conducted at the same test facility
- Rationale for selecting satellite groups: 5 animals/sex/group were selected for functional observations, locomotor activitiy, clinical pathology, macroscopic exmination, organ weights and histopathology
- Section schedule rationale (if not random):
Females which delivered: PND 14-16
Females that failed to deliver: Post-coitum Days 25-28 (females with evidence of mating) or approximately 25 days after the last day of the mating period (female without evidence of mating).
Females with total litter loss: within 24 hours of litter loss
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
at least twice daily for mortality and viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
at least once daily. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

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

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

WATER CONSUMPTION:
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on PND 14-16
- Organs examined: full post-mortem necropsy, with special attention paid to the reproductive organs.

OTHER:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to the scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters examined: WBC, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), RBC, reticulocytes, RDW, haemoglobin, haematocrit, MCV, MCY, MCHC, platelets; prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled necropsy
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters: ALAT, ASAT, ALP, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: the selected males during week 4 of treatment; the selected females during the last week of lactation (PND 6-13).
- Dose groups that were examined: all, on selected 5 animals/sex/dose
- Battery of functions tested: sensory activity (hearing ability, pupillary reflex, static righting reflex), grip strength (fore- and hind-limbs), motor activity (total movement and ambulations)

HISTOPATHOLOGY: Yes
- On selected 5 females/group in control and high-dose groups and all animals that were euthanized in extremis: adrenal glands, brain (cerebellum, mid-brain, cortex), caecum, cervix, clitoral gland, colon, duodenum, eyes (with optic nerve if detectable and Harderian gland), mammary gland area, femur including joint, heart, ileum, jejunum, kidneys, liver, lung, lymph nodes (mandibular, mesenteric), ovaries, Peyers' patches if detectable, pituitary gland, preputial gland, rectum, sciatic nerve, skeletal muscles, spinal cord (cervica, midthoracic, lumbar), spleen, sternum with bone marrox, stomach, thymus, thyroid including parathyroid if detectable, trachea, urinary bladder, uterus, vagina, all gross lesions.
- On all animals: all gross lesions
- All selected females of low- and mid-dose groups: liver, urinary bladder, based on possible effects on these organs in the high-dose group

ORGAN WEIGHTS: Yes
The following organ weights were recorded:
- On selected 5 females/group: adrenal glands, brain, heart, kidneys, liver, ovaries, spleen, thymus, thyroid including parathyroid if detectable, uterus including cervix.
- All remaining animals: thyroid
Ovaries and uterine content:
The ovaries and uterine content was 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:
Examinations were conducted on the pups, as females were allowed to litter naturally (pups found dead, at culling and at a final sacrifice on PND 13-15)
- External examinations: Yes, all per litter
- Soft tissue examinations: no specific examinations were conducted, but gross necropsy was conducted on all pups
- Skeletal examinations: no specific examinations were conducted, but gross necropsy was conducted on all pups
- Head examinations: no specific examinations were conducted, but gross necropsy was conducted on all pups
Statistics:
The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data
• The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Indices:
The following reproductive indices were calculated:
The following indices were calculated:
Mating index = 100% x (number of females mated/number of females paired)
Precoital time = number of days between initiation of cohabitation and confirmation of mating
Fertility index = 100% x (number of pregnant females/number of females paired)
Conception index = 100% x (number of pregnant females/number of females mated)
Gestation index = 100% x (number of females bearing live pups/number of pregnant females)
Duration of gestation = number of days between confirmation of mating and the beginning of parturition
The following offspring indices were calculated:
Post-implantation survival index = 100% x (total number of offspring born/total number of uterine implantation sites)
Live birth index = 100% x (number of live offspring on day 1 after littering/total number of offspring born)
Percentage live males at first litter check = 100% x (number of live male pups at first litter check/number of live pups at first litter check)
Percentage live females at first litter check = 100% x (number of live female pups at first litter check/number of live pups at first litter check)
Viability index = 100% x (number of live osffspring on PND 4 before culling/number of live offspring on PND 1 after littering)
Lactation index = 100% x (numebr of live offspring on PND 13 after littering/number of live offspring on PND 4 after culling)
Historical control data:
Historical control data from the same test laboratory are available for a period of 2015-2017.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period.
Salivation seen in a dose-related incidence after dosing among animals of the control and treated groups was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). For the test item treated groups, this sign may be related to taste of the test item.
All other clinical signs among surviving animals occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were not considered to be signs of toxicological relevance. No findings were noted during the arena observations in this study.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test item.
One female at 750 mg/kg bw/day was sacrificed on PND 1 due to total litter loss.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of maternal animals in all treatment groups remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was not considered affected by treatment.
The statistically significant higher relative food intake of females at 750 mg/kg bw/day over Days 11-14 of the post-coitum period was considered unrelated to treatment, since this change was slight and not consistently present with continuing treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day, the following (statistically significant) changes in haematology parameters distinguished treated animals from control animals:
- Lower mean corpuscular volume (MCV) in females.
- Lower mean corpuscular haemoglobin (MCH) in females.
The statistically significant lower red cell distribution width of females at 250 mg/kg bw/day was not considered to be related to treatment as this occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher alanine aminotransferase activity (ALAT) in females at 750 mg/kg bw/day (not statistically significant; higher mean primarily due to one animal).
- Higher aspartate aminotransferase activity (ASAT) in females at 750 mg/kg bw/day (not statistically significant; higher mean primarily due to one animal).
- Higher total bilirubin in one female at 750 mg/kg bw/day (not statistically significant)
- Higher bile acids in three males and in one female at 750 mg/kg bw/day (not statistically significant).
- Higher sodium in females at 750 mg/kg bw/day.
Any other statistically significant changes in clinical biochemistry parameters were not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day, females had a statistically significantly lower motor activity for both total movements and ambulations. At 250 mg/kg bw/day, motor activity (total movements only) was statistically significantly higher than controls.
All groups showed a similar habituation profile in motor activity, with high activity in the first interval with a decreasing trend in activity over the duration of the test period. Other functional observation parameters were not considered to be affected by treatment. Hearing ability, pupillary reflex and static righting reflex were normal in all selected animals. Fore- and hindlimb grip strength was similar between control and treated groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant test item-related higher organ weights were observed at 750 mg/kg bw/day in liver. Test item-related higher liver weights (absolute and relative to body weights) were noted in the 750 mg/kg bw/day group females (relative to body weight 21% higher than in controls, p < 0.01).
Any other differences, including those that reached statistical significance were considered not to be treatment-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related macroscopic findings were noted in maternal animals. The recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with the test substance were noted starting at 250 mg/kg bw/day in the liver and at 750 mg/kg bw/day in the urinary bladder of female animals.
In the liver, bile duct hypertrophy/hyperplasia was observed starting at 250 mg/kg bw/day in females (up to marked). Peribiliary inflammatory cell infiltrate was observed starting at 250 mg/kg bw/day in females (up to slight). Coagulative necrosis was observed at 750 mg/kg bw/day in 2 females (up to slight). Peribiliary fibrosis was observed at 750 mg/kg bw/day in all 5 females (up to slight).
In the urinary bladder, urothelium hyperplasia was observed at increased incidence and severity at 750 mg/kg in all females (up to slight). The hyperplasia was in general diffuse and without cellular atypia.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Estrous cycle:
At 750 mg/kg bw/day, two females at 750 mg/kg bw/day were acyclic; one of these females did not mate and another one had a normal litter. Acyclic estrous cycles are infrequent occurrences in this type of study (1/316 historical control dams, period 2015-2017).
Length and regularity of the estrous cycle at 75 and 250 mg/kg bw/day were not considered to have been affected by treatment. Most females had regular cycles of 4 days. Extended di-estrus occurred in two females at 750 mg/kg bw/day with a regular cycle. An irregular cycle was also noted for one female at 750 mg/kg bw/day with normal litter. Given their incidental nature and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment. However, in combination with elongated precoital time in two other females at 750 mg/kg bw/day and developmental effects these findings may indicate adverse effect on reproduction (see cross-references).
Number of abortions:
no effects observed
Description (incidence and severity):
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day, the total number of offspring born compared to the total number of uterine implantations was lower than controls (67% vs. 93% in the control group). At 75 and 250 mg/kg bw/day, post-implantation survival index was considered unaffected by treatment.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment.
Four pups at 250 mg/kg bw/day (from two litters), and eight pups at 750 mg/kg bw/day (all from one litter, total litter loss) were found dead or missing at first litter check. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since this incidence remained within the range considered normal for pups of this age and since total litter loss can occasionally occur in this type of study.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Duration of gestation was not considered to be affected by treatment.
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
A total of two control females and one female at 75 mg/kg bw/day were not pregnant. Since these cases of non-pregnancy showed no dose-related incidence across the dose groups, this was not considered to be related to treatment. The isolated occurrences of two cases of non-pregnancy at 750 mg/kg bw/day were within the range normally encountered in this type of study. However, based on other developmental effects at this dose level, it could not be excluded that this was related to treatment
Other effects:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day, two females were acyclic. Two other females at this dose had an elongated precoital time of 12 days. Both occurrence of acyclic estrous cycles and a precoital time of 12 days are infrequent occurrences in this type of study. In combination with the observed developmental effects (see the cross reference), these occurrences were considered to represent an adverse effect on reproduction at 750 mg/kg bw/day.
Mating index was not affected by treatment. One female at 750 mg/kg bw/day showed no evidence of mating. This was due to the fact that this female was separated from her male prematurely as this female was incorrectly recorded as having shown evidence of mating. All other females in this study showed evidence of mating.
No signs of difficult or prolonged parturition were noted among the pregnant females. No deficiencies in maternal care were observed.
Key result
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: liver
Fetal body weight changes:
not examined
Description (incidence and severity):
Not applicable, females were allowed to litter naturally
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment.
Four pups at 250 mg/kg bw/day (from two litters) and eight pups at 750 mg/kg bw/day (total litter loss) were found dead or missing at first litter check. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since this incidence remained within the range considered normal for pups of this age and since total litter loss can occasionally occur in this type of study.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was not considered to be affected by the treatment.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Litter size at 750 mg/kg bw/day was lower than the control group at this dose (6.6 vs. 11.0 in the control group).
Body weights of pups were not considered to be affected by treatment.
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
At 250 mg/kg bw/day, a higher postnatal loss was recorded (a total of nine pups were found missing over 5/10 litters). This incidence was considered higher than normally encountered in this type of study. At 750 mg/kg bw/day, no higher postnatal loss was recorded. However, it is conceivable that at 250 mg/kg bw/day pup death occurred at a later stage during development than at 750 mg/kg where post-implantation loss (i.e. earlier during development) may have resulted in a lower number of pups (lower litter size and lower total number of litters). This lower number of pups may have compromised the (statistical) chance of detecting increased pup loss as observed at 250 mg/kg bw/day, and could explain the absence of higher postnatal loss at 750 mg/kg bw/day.
External malformations:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore not considered to be related to treatment.
Skeletal malformations:
not examined
Description (incidence and severity):
No specific examinations were performed, but no macroscopic findings were noted among pups that were considered to be related to treatment.
Visceral malformations:
not examined
Description (incidence and severity):
No specific examinations were performed, but no macroscopic findings were noted among pups that were considered to be related to treatment.
Other effects:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment. The nature and incidence of clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be related to treatment.
Treatment up to and including 750 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13. No toxicologically relevant changes in anogenital distance (absolute and normalized for body weight) were recorded for male and female pups. The statistically significantly higher mean normalized anogenital distance of male pups at 75 mg/kg bw/day and female pups at 750 mg/kg bw/day were not considered toxicologically relevant since means remained well within the range considered normal for rats of this age and strain.
Details on embryotoxic / teratogenic effects:
At 750 mg/kg bw/day, a limited number of litters was available for evaluation; a total of 6/10 females delivered litters that survived beyond PND 1. The other four females did not deliver (healthy) offspring, and for three of these females this was due to total litter loss or non-pregnancy. For one other female at this dose, mating was inadvertently discontinued and she had no offspring accordingly. In isolation, absence of offspring for three females can occur as background findings in this type of study. However, given the combined occurrence of these findings in the high dose group along with other developmental effects, this was considered to represent an adverse developmental effect at 750 mg/kg bw/day.
At 750 mg/kg, a lower post-implantation survival index (i.e. total number of offspring born compared to the total number of uterine implantations) was recorded (67% vs. 93% in the control group). Also, litter size was lower than the control group at this dose (6.6 vs. 11.0 in the control group). These represented adverse developmental effects.
At 250 mg/kg bw/day, a higher postnatal loss was recorded (a total of nine pups were found missing over 5/10 litters). This incidence was considered higher than normally encountered in this type of study. At 750 mg/kg bw/day, no higher postnatal loss was recorded. However, it is conceivable that at 250 mg/kg bw/day pup death occurred at a later stage during development than at 750 mg/kg where post-implantation loss (i.e. earlier during development) may have resulted in a lower number of pups (lower litter size and lower total number of litters). This lower number of pups may have compromised the (statistical) chance of detecting increased pup loss as observed at 250 mg/kg bw/day, and could explain the absence of higher postnatal loss at 750 mg/kg bw/day. It was therefore considered that the higher postnatal loss at 250 mg/kg bw/day represented an adverse developmental effect.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in postnatal survival
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Analytical determination of the test formulations:

Mean accuracies in the low-, mid- and high-dose groups were 100, 11 and 108%, respectively (n = 6, 2, 6). For the mid-dose formulation prepared prior to treatment, the mean accuracy was above the

target concentration (i.e. 111% of target). Since this value only marginally exceeded the range of 90-110% of nominal, it was considered acceptable for the purpose of this study.

The coefficient of variation for low- and high-dose groups was 16 and 3.0%, respectively. ≤ 10%). The criterion for homogeneity is that the coefficient of variation ≤10%. For the low-dose formulation prepared prior to treatment, the coefficient of variation was above the target concentration (i.e. 16% of target). This was mainly due to two lower accuracy values (79 and 80%) at 90% sampling height. Overall, these results were considered acceptable for the purpose of this study.

Conclusions:
In the GLP-compliant OECD guideline 422 study with rats that received the test substance in corn oil at dose levels of 0, 75, 250 and 750 mg/kg bw/day, at the highest dose level of 750 mg/kg bw/day a lower post-implantation survival index was recorded (67% vs. 93% in the control group), and litter size was lower than the control group at this dose (6.6 vs. 11.0 in the control group). At 250 mg/kg bw/day, 9 pups went missing during PND 1-4. Furthermore, two females were not pregnant and one had a total litter loss at 750 mg/kg bw/day. These effects are considered to represent an adverse effect on development. As a result, the NOAEL for developmental toxicity was set at 75 mg/kg bw/day.
General adverse toxic effects on the liver (hypertrophy/hyperplasia of the bile ducts and peribiliary inflammatory cell infiltrates) were observed starting from the dose level of 250 mg/kg bw/day in females and increased in severity and incidence at 750 mg/kg bw/day. At 750 mg/kg bw/day they were also accompanied by (not statistically significant) changes in clinical chemistry (higher ALAT and ASAT activities, higher bile acids and bilirubin levels) and liver weight changes. Based on these findings the NOAEL for maternal toxicity was set at 75 mg/kg bw/day.
Executive summary:

In the GLP-compliant OECD guideline 422 study, groups of 10 rats/sex/dose were treated with the test item in corn oil at dose levels of 0 (vehicle controls), 75, 250 and 750 mg/kg bw/day 2 weeks before mating, during mating and during gestation and lactation until PND 14-16 (females that delivered), for a minimum of 28 days (males) or 42 -56 days (females). At 750 mg/kg bw/day, a lower post-implantation survival index was recorded (67% vs. 93% in the control group) together with the lower litter size (6.6 vs. 11.0 in the control group). in addition, two females were not pregnant and one had a total litter loss at this dose level. Furthermore, at 250 mg/kg bw/day 9 pups went missing between PND 1 -4. This incidence was considered higher than normally encountered in this type of study. At 750 mg/kg bw/day, no higher postnatal loss was recorded. However, it is conceivable that at 250 mg/kg bw/day pup death occurred at a later stage during development than at 750 mg/kg bw/day where post-implantation loss (i.e. earlier during development) may have resulted in a lower number of pups (lower litter size and lower total number of litters). Therefore the NOAEL for developmental effects was set at 75 mg/kg bw/day.

Adverse effects on the liver (hypertrophy/hyperplasia of the bile ducts and peribiliary inflammatory cell infiltrates) were observed starting from the dose level of 250 mg/kg bw/day in maternal animals and increased in severity and incidence at 750 mg/kg bw/day. At 750 mg/kg bw/day they were also accompanied by (not statistically significant) changes in clinical chemistry (higher ALAT and ASAT activities, higher bile acids and bilirubin levels) and statistically significantly increased absolute and relative liver weights. Furthermore, coagulative necrosis was observed at 750 mg/kg bw/day in 2 females (up to slight) and peribiliary fibrosis was observed in all 5 females (up to slight). At 750 mg/kg bw/day urothelium hyperplasia was observed at increased incidence and severity in all females up to slight degree. Although the hyperplasia was in general diffuse and without cellular atypia, based on the proliferative character this finding was regarded as adverse. Based on the observed effects, the NOAEL for general parental toxicity was set at 75 mg/kg bw/day.

The treatment at the highest dose level of 750 mg/kg bw/day had an adverse effect on the oestrous cycle, precoital time and the total number of pregnant. Two females in the high-dose group were acyclic and two other had a longer precoital time in comparison with controls. Both occurrence of acyclic estrous cycles and a precoital time of 12 days are infrequent occurrences in this type of study. In combination with observed developmental effects they are considered to represent adverse effects on fertility. Therefore the NOAEL for reproductive toxicity was set at 250 mg/kg bw/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Test material form:
solid
Details on test material:
- Appearance: dark red-brown sollid
- Storage conditions: at room temperature
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stability in corn oil for at least 6 hours at room temperature is confirmed over the concentration range 1 to 200 mg/mL Stability in corn oil for at least 8 days in the refrigerator is confirmed for the concentration 200 mg/mL.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was heated up to a maximum temperature of 69 ºC for a maximum of 2 hours and 27 minutes before formulating.

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Remarks:
outbred, SPF-quality
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males ca. 10 weeks, females ca. 13 weeks (at the start of F0 treatment)
- Weight at study initiation: males 277-302 g, females 201-239 g
- Fasting period before study: none
- Housing: sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied.
At pretest: females were housed in groups of 5/cage in Macrolon type IV plastic cages
Pre-mating: in groups of 5/sex/dose in Macrolon type IV plastic cages
Mating: on 1:1 basis m:f in Macrolon type III plastic cages
Post-mating: males in their home cages (5/dose), females individually in Macrolon type III plastic cages
Lactation: females were housed with pups in Macrolon type III cages. During locomotor activity measurements pups were kep warm in their home cages using warm water bottles. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
Locomotor activity monitoring: individually in a Hi-temp cages without cage enrichment, bedding material, food and water
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum, except during motor activity measurements for max. 2 hours and overnight before terminal sacrifice
- Water: tap water, ad libitum, except during motor activity measurements for max. 2 hours
- Acclimation period: at least 5 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item was heated up to a maximum temperature of 69 ºC for a maximum of 2 hours and 27 minutes before formulating. High-dose formulations (w/w) were heated to 69 °C for a maximum duration of 5 hours and 40 minutes under continuous stirring, until a visually homogenous formulation was obtained. After cooling down, these formulations were stored in the refrigerator for at least an overnight period, but maximally 8 days. Low- and mid-dose formulations (w/w) were prepared by diluting the high-dose formulation with the required amount of vehicle daily within 6 hours prior to dosing, and were homogenized to a visually acceptable level. High-dose formulation was acclimated to room temperature conditions and made visually homogenous prior to this dilution.
Adjustment was made for specific gravity of the vehicle. Density of the high-dose formulation was determined on a single occasion during the treatment period (0.93 g/mL), after the formulation had been acclimatized to room temperature conditions on the day of preparation of the low- and mid-dose concentrations. Until then a density value of 0.92 g/mL was taken for the high-dose formulation (i.e. the same density as for the vehicle). No correction was made for the purity/composition of the test item.

- VEHICLE
- Justification for use and choice of vehicle (if other than water):
based on trial formulations performed at Charles River Den Bosch
- Amount of vehicle (if gavage): 5 mL/kg bw
- Adjustment was made for specific gravity of the vehicle (0.92 g/mL).
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: a maximum of 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged individually in Macrolon type III plastic cage with sawdust as bedding material
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during pretreatment phase (15 November 2016) according to a validated method (UPLC-MS). Samples were collected and analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Duration of treatment / exposure:
Males: 29 days (2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy)
Females that delivered: 50-56 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy
Females which failed to deliver healthy offspring: 42-53 days
Pups were not treated, but could have been exposed to the test substance via lactation.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose
Details on study schedule:
- Age at mating of the mated animals in the study: males ca. 12 weeks, females ca. 15 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle controls (Group 1)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results of a 10-day dose range finding study conducted at the same test facility
- Rationale for selecting satellite groups: 5 animals/sex/group were selected for functional observations, locomotor activitiy, clinical pathology, macroscopic exmination, organ weights and histopathology
- Section schedule rationale (if not random):
Males: following the completion of the mating period (a minimum of 28 days test substance administration)
Females which delivered: PND 14-16
Females that failed to deliver: Post-coitum Days 25-28 (females with evidence of mating) or approximately 25 days after the last day of the mating period (female without evidence of mating).
Females with total litter loss: within 24 hours of litter loss
Positive control:
Not applicable

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
at least once daily. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

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

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

WATER CONSUMPTION:
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced

OTHER:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to the scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters examined: WBC, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), RBC, reticulocytes, RDW, haemoglobin, haematocrit, MCV, MCY, MCHC, platelets; prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled necropsy
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters: ALAT, ASAT, ALP, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: the selected males during week 4 of treatment; the selected females during the last week of lactation (PND 6-13).
- Dose groups that were examined: all, on selected 5 animals/sex/dose
- Battery of functions tested: sensory activity (hearing ability, pupillary reflex, static righting reflex), grip strength (fore- and hind-limbs), motor activity (total movement and ambulations)

Thyroid hormone measurements:
- Serum T4 concentration was measured in males prior to scheduled sacrifice
Oestrous cyclicity (parental animals):
Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymis weight, staging of spermatogenesis (PAS/haematoxylin staining of the testes slides)
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); excess pups were killed and discarded. Blood samples were collected from two of the surplus pups for the thyroid hormone analysis.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number of live and dead pups (on PND 1 and daily thereafter), sex of pups (PND 1 and 4), stillbirths, live births, postnatal mortality, presence of gross anomalies, body weights (PND 1, 4, 7, 13 on live pups), physical or behavioural abnormalities, anogenital distance (AGD) (PND 1 on all live pups), presence of nipples/areolae in male pups (PND 13)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead. The stomach of pups not surviving to the scheduled necropsy date was examined for the
presence of milk, if possible.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: not performed

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: not performed
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after mating was completed, with a minimum of 28 days of treatment
- Females which delivered: PND 14-16
- Females that failed to deliver: Post-coitum Days 25-28 (females with evidence of mating) or approximately 25 days after the last day of the mating period (female without evidence of mating).
- Females with total litter loss: within 24 hours of litter loss

GROSS NECROPSY: Yes
Full post-mortem necropsy, with special attention paid to the reproductive organs.

HISTOPATHOLOGY: Yes
- On selected 5 animals/sex/group in control and high-dose groups and all animals that were euthanized in extremis: adrenal glands, brain (cerebellum, mid-brain, cortex), caecum, cervix, clitoral gland, colon, coagulation gland, duodenum, epididymides, eyes (with optic nerve if detectable and Harderian gland), mammary gland area (males and females), femur including joint, heart, ileum, jejunum, kidneys, liver, lung, lymph nodes (mandibular, mesenteric), ovaries, Peyers' patches if detectable, pituitary gland, preputial gland, prostate gland, rectum, sciatic nerve, seminal vesicles, skeletal muscles, spinal cord (cervica, midthoracic, lumbar), spleen, sternum with bone marrox, stomach, testes, thymus, thyroid including parathyroid if detectable, trachea, urinary bladder, uterus, vagina, all gross lesions.
- On selected 5 males in all groups and all males that failed to sire: additional testes slides
- On all animals: all gross lesions
- All selected males of low- and mid-dose groups: thymus, spleen and sternal bone marrow, based on possible effects on these organs in the high-dose group
- All selected males and females of low- and mid-dose groups: liver, urinary bladder, based on possible effects on these organs in the high-dose group

ORGAN WEIGHTS: Yes
The following organ weights were recorded:
- On selected 5 animals/sex/group: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostates, seminal vesicles including coagulating glands, spleen, testes, thymus, thyroid including parathyroid if detectable, uterus including cervix.
- All remaining animals: epididymides, prostate, seminal vesicles including coagulation glands, testes, thyroid
Postmortem examinations (offspring):
SACRIFICE
- PND 4 (culling); PND 13-15 (terminal sacrifice)
- These animals were subjected to postmortem examinations. All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGTHS: not performed.

THYROID HORMONE ANALYSIS:
On PND 4 (at culling), from 2 surplus pups per litter, blood samples were collected for possible T4 analysis (the analysis was not performed, as no effects were seen on T4 in pups on PND 13-15). On PND13-15, blood was collected fro 2 pups/litter for T4 analysis and possible TSH analysis (TSH analysis was not performed, as no effects were seen on the T4 concentration).
Statistics:
The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data
• The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Reproductive indices:
The following indices were calculated:
Mating index = 100% x (number of females mated/number of females paired)
Precoital time = number of days between initiation of cohabitation and confirmation of mating
Fertility index = 100% x (number of pregnant females/number of females paired)
Conception index = 100% x (number of pregnant females/number of females mated)
Gestation index = 100% x (number of females bearing live pups/number of pregnant females)
Duration of gestation = number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Post-implantation survival index = 100% x (total number of offspring born/total number of uterine implantation sites)
Live birth index = 100% x (number of live offspring on day 1 after littering/total number of offspring born)
Percentage live males at first litter check = 100% x (number of live male pups at first litter check/number of live pups at first litter check)
Percentage live females at first litter check = 100% x (number of live female pups at first litter check/number of live pups at first litter check)
Viability index = 100% x (number of live osffspring on PND 4 before culling/number of live offspring on PND 1 after littering)
Lactation index = 100% x (numebr of live offspring on PND 13 after littering/number of live offspring on PND 4 after culling)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period.
Salivation seen in a dose-related incidence after dosing among animals of the control and treated groups was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). For the test item treated groups, this sign may be related to taste of the test item.
All other clinical signs among surviving animals occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were not considered to be signs of toxicological relevance. No findings were noted during the arena observations in this study.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test item.
One control male was sacrificed in extremis on Day 28 due to a serious eye injury. At clinical observation, the left eye appeared exophthalmic and dehydrated and was discolored black. Additionally, the right cheek was thickened. At necropsy, the left eye was recorded as desiccated, with marked acute necrotizing inflammation as microscopic correlate.
One female at 750 mg/kg bw/day was sacrificed on PND 1 due to total litter loss.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day, males had a slightly lower weight gain throughout the treatment period, achieving a level of statistical significance on Day 8 of the premating period only. Mean body weight at the end of treatment were approximately 5% lower than the control group.
Body weights and body weight gain of other treated groups remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was not considered affected by treatment.
The statistically significant higher relative food intake of females at 750 mg/kg bw/day over Days 11-14 of the post-coitum period was considered unrelated to treatment, since this change was slight and not consistently present with continuing treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day, the following (statistically significant) changes in haematology parameters distinguished treated animals from control animals:
- Lower relative lymphocyte counts in males (not statistically significant).
- Higher relative monocyte counts in males.
- Higher relative eosinophil counts in males.
- Lower red blood cell counts in males.
- Higher reticulocyte counts in males (not statistically significant), primarily attributed to a high value of one male
- Lower haemoglobin in males.
- Lower haematocrit in males.
- Lower mean corpuscular volume (MCV) in females.
- Lower mean corpuscular haemoglobin (MCH) in females.
The statistically non-significant lower activated partial thromboplastin time of males at 750 mg/kg bw/day was not considered to be of toxicological relevance since the opposite effect (i.e. an increase) would be expected in case of target organ toxicity.
The statistically significant lower red cell distribution width of females at 250 mg/kg bw/day was not considered to be related to treatment as this occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher alanine aminotransferase activity (ALAT) in males at 250 mg/kg bw/day (not statistically significant) and in males and females at 750 mg/kg bw/day (not statistically significant for females; higher mean primarily due to one animal).
- Higher aspartate aminotransferase activity (ASAT) in males at 250 mg/kg bw/day (not statistically significant; primarily due to one animal) and in males and females at 750 mg/kg bw/day (not statistically significant for females; higher mean primarily due to one animal).
- Higher alkaline phosphatase activity (ALP) in males at 750 mg/kg bw/day
- Higher total protein in males at 750 mg/kg bw/day
- Higher total bilirubin in males at 750 mg/kg bw/day (not statistically significant; higher mean primarily due to one male) and in one female at 750 mg/kg bw/day (not statistically significant)
- Higher urea in males at 750 mg/kg bw/day.
- Higher creatinine in males at 750 mg/kg bw/day.
- Higher cholesterol in males at 750 mg/kg bw/day.
- Higher bile acids in three males and in one female at 750 mg/kg bw/day (not statistically significant for both sexes).
- Higher sodium in females at 750 mg/kg bw/day.
- Lower chloride in males at 750 mg/kg bw/day
Any other statistically significant changes in clinical biochemistry parameters were not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.
Serum levels of T4 in F0 males were not considered to be affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day, females had a statistically significantly lower motor activity for both total movements and ambulations. At 250 mg/kg bw/day, motor activity (total movements only) was statistically significantly higher than controls.
All groups showed a similar habituation profile in motor activity, with high activity in the first interval with a decreasing trend in activity over the duration of the test period. Other functional observation parameters were not considered to be affected by treatment. Hearing ability, pupillary reflex and static righting reflex were normal in all selected animals. Fore- and hindlimb grip strength was similar between control and treated groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with the test substance were noted starting at 250 mg/kg bw/day in the liver of both sexes and spleen of males, and at 750 mg/kg bw/day in the urinary bladder of both sexes and thymus of males.
In the liver, bile duct hypertrophy/hyperplasia was observed starting at 250 mg/kg bw/day in males (up to moderate) and females (up to marked). Peribiliary inflammatory cell infiltrate was observed starting at 250 mg/kg bw/day in males (up to marked) and females (up to slight). Coagulative necrosis was observed starting at 250 mg/kg bw/day in one male (minimal) and in 4 males at 750 mg/kg bw/day (up to moderate) and at 750 mg/kg bw/day in 2 females (up to slight). Peribiliary fibrosis was observed at 750 mg/kg bw/day in all 5 males (up to moderate) and all 5 females (up to slight).
In the urinary bladder, urothelium hyperplasia was observed at increased incidence and severity at 750 mg/kg in all males (up to slight) and all females (up to slight). The hyperplasia was in general diffuse and without cellular atypia.
In thymus, lymphoid atrophy and increased lymphocytosis were observed in 750 mg/kg bw/day males (minimal).
In the spleen, hematopoiesis was observed at increased incidence and severity in all males starting at 250 mg/kg bw/day (up to moderate).
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
Estrous cycle:
At 750 mg/kg bw/day, two females at 750 mg/kg bw/day were acyclic; one of these females did not mate and another one had a normal litter. Acyclic estrous cycles are infrequent occurrences in this type of study (1/316 historical control dams, period 2015-2017).
Length and regularity of the estrous cycle at 75 and 250 mg/kg bw/day were not considered to have been affected by treatment. Most females had regular cycles of 4 days. Extended di-estrus occurred in two females at 750 mg/kg bw/day with a regular cycle. An irregular cycle was also noted for one female at 750 mg/kg bw/day with normal litter. Given their incidental nature and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Spermatogenic staging profiles were normal for all males examined. There were no effects on the testes and epididymides weights. No further examinations were performed.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day, two females were acyclic. Two other females at this dose had an elongated precoital time of 12 days. Both occurrence of acyclic estrous cycles and a precoital time of 12 days are infrequent occurrences in this type of study. In combination with the observed developmental effects (see the cross reference), these occurrences were considered to represent an adverse effect on reproduction at 750 mg/kg bw/day. Furthermore, two females at 750 mg/kg bw/day were not pregnant. The isolated occurrences of non-pregnancy at 750 mg/kg bw/day were within the range normally encountered in this type of study. However, based on other developmental effects at this dose level, it could not be excluded that this was related to treatment.
At 750 mg/kg bw/day, the total number of offspring born compared to the total number of uterine implantations was lower than controls (67% vs. 93% in the control group). At 75 and 250 mg/kg bw/day, post-implantation survival index was considered unaffected by treatment.
One female at 250 mg/kg bw/day had implantation sites only and one female at 750 mg/kg bw/day had total litter loss on PND 1. The isolated occurrence of total litter loss at 750 mg/kg bw/day was within the range normally encountered in this type of study. However, based on other developmental effects at this dose level, it could not be excluded that this occurrence was related to treatment
No reproduction toxicity was observed at 75 and 250 mg/kg bw/day. No treatment-related changes were noted in any of the other reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, and number of implantations, spermatogenic profiling, and histopathological examination of reproductive organs). Gestation index and pregnancy duration were not considered to be affected by the treatment. No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive performance
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
75 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 (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
750 mg/kg bw/day (actual dose received)
System:
female reproductive system
Organ:
other: not applicable, effects on oestrous cycle and number of pregnant females and females with a total litter loss
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment. The nature and incidence of clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be related to treatment.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment.
Four pups at 250 mg/kg bw/day (from two litters) and eight pups at 750 mg/kg bw/day (total litter loss) were found dead or missing at first litter check. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since this incidence remained within the range considered normal for pups of this age and since total litter loss can occasionally occur in this type of study.
At 250 mg/kg bw/day, a total of nine pups were found missing between PND 1 and 4 (out of 5 litters), resulting in a statistically significant higher postnatal loss. Although the mean viability index did not show a dose-related trend across the dose groups, the number of pups missing at 250 mg/kg bw/day was considered to exceed the range normally encountered in this type of study.
At 75 and 750 mg/kg bw/day, the number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered affected by treatment. One pup of the control group, one pup at 75 mg/kg bw/day, and one pup at 750 mg/kg bw/day were found missing and were most likely cannibalised. No toxicological relevance was attributed to these missing pups as the incidence was considered to remain within the range considered normal for this type of study.
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not considered to be affected by treatment. No pups were found dead/missing between lactation Days 5 and 13.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were not considered to be affected by treatment.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 13-15 pups were not considered to be affected by treatment. No other clinical chemistry parameters were examined.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore not considered to be related to treatment.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Treatment up to and including 750 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13. No toxicologically relevant changes in anogenital distance (absolute and normalized for body weight) were recorded for male and female pups. The statistically significantly higher mean normalized anogenital distance of male pups at 75 mg/kg bw/day and female pups at 750 mg/kg bw/day were not considered toxicologically relevant since means remained well within the range considered normal for rats of this age and strain. Sex ratio was not considered to be affected by the treatment.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

At 750 mg/kg bw/day, a limited number of litters was available for evaluation; a total of 6/10 females delivered litters that survived beyond PND 1. The other four females did not deliver (healthy) offspring, and for three of these females this was due to total litter loss or non-pregnancy. For one other female at this dose, mating was inadvertently discontinued and she had no offspring accordingly. In isolation, absence of offspring for three females can occur as background findings in this type of study. However, given the combined occurrence of these findings in the high dose group along with other developmental effects, this was considered to represent an adverse developmental effect at 750 mg/kg bw/day.
At 750 mg/kg, a lower post-implantation survival index (i.e. total number of offspring born compared to the total number of uterine implantations) was recorded (67% vs. 93% in the control group). Also, litter size was lower than the control group at this dose (6.6 vs. 11.0 in the control group). These represented adverse developmental effects.
At 250 mg/kg bw/day, a higher postnatal loss was recorded (a total of nine pups were found missing over 5/10 litters). This incidence was considered higher than normally encountered in this type of study. At 750 mg/kg bw/day, no higher postnatal loss was recorded. However, it is conceivable that at 250 mg/kg bw/day pup death occurred at a later stage during development than at 750 mg/kg where post-implantation loss (i.e. earlier during development) may have resulted in a lower number of pups (lower litter size and lower total number of litters). This lower number of pups may have compromised the (statistical) chance of detecting increased pup loss as observed at 250 mg/kg bw/day, and could explain the absence of higher postnatal loss at 750 mg/kg bw/day. It was therefore considered that the higher postnatal loss at 250 mg/kg bw/day represented an adverse developmental effect.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality

Target system / organ toxicity (F1)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
System:
other: not applicable, higher mortality and post-implantation loss
Organ:
other: not applicable, higher mortality and post-implantation loss
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Analytical determination of the test formulations:

Mean accuracies in the low-, mid- and high-dose groups were 100, 11 and 108%, respectively (n = 6, 2, 6). For the mid-dose formulation prepared prior to treatment, the mean accuracy was above the

target concentration (i.e. 111% of target). Since this value only marginally exceeded the range of 90-110% of nominal, it was considered acceptable for the purpose of this study.

The coefficient of variation for low- and high-dose groups was 16 and 3.0%, respectively. ≤ 10%). The criterion for homogeneity is that the coefficient of variation ≤10%. For the low-dose formulation prepared prior to treatment, the coefficient of variation was above the target concentration (i.e. 16% of target). This was mainly due to two lower accuracy values (79 and 80%) at 90% sampling height. Overall, these results were considered acceptable for the purpose of this study.

Applicant's summary and conclusion

Conclusions:
In the GLP-compliant OECD guideline 422 study with rats that received the test substance in corn oil at dose levels of 0, 75, 250 and 750 mg/kg bw/day, two females in the high-dose group were acyclic and two other had a longer precoital time in comparison with controls. In addition, two females were not pregnant and one had a total litter loss. These effects are considered to represent an adverse effect on reproduction. The NOAEL for reproductive toxicity was therefore set at 250 mg/kg bw/day.
At 750 mg/kg bw/day a lower post-implantation survival index was recorded (67% vs. 93% in the control group), and litter size was lower than the control group at this dose (6.6 vs. 11.0 in the control group) at this dose level. At 250 mg/kg bw/day, 9 pups went missing during PND 1-4. These effects are considered to represent an adverse effect on development. As a result, the NOAEL for developmental toxicity was set at 75 mg/kg bw/day.
General adverse toxic effects on the liver (hypertrophy/hyperplasia of the bile ducts, peribiliary inflammatory cell infiltrates and focal coagulative necrosis) were observed starting from the dose level of 250 mg/kg bw/day and increased in severity and incidence at 750 mg/kg bw/day. At 750 mg/kg bw/day they were also accompanied by changes in clinical chemistry (higher ALAT and ASAT activities, higher bile acids and bilirubin levels, statistically significant in males) and liver weight changes. Furthermore, coagulative necrosis was observed starting in 4 males at 750 mg/kg bw/day (up to moderate) and at 750 mg/kg bw/day in 2 females (up to slight). Peribiliary fibrosis was observed at 750 mg/kg bw/day in all 5 males (up to moderate) and all 5 females (up to slight). Based on these findings the NOAEL for general toxicity was set at 75 mg/kg bw/day.
Executive summary:

In the GLP-compliant OECD guideline 422 study, groups of 10 rats/sex/dose were treated with the test item in corn oil at dose levels of 0 (vehicle controls), 75, 250 and 750 mg/kg bw/day 2 weeks before mating, during mating and during gestation and lactation until PND 14-16 (females that delivered), for a minimum of 28 days (males) or 42 -56 days (females). Adverse effects on the liver (hypertrophy/hyperplasia of the bile ducts, peribiliary inflammatory cell infiltrates and focal coagulative necrosis (minimal in one male)) were observed starting from the dose level of 250 mg/kg bw/day and increased in severity and incidence at 750 mg/kg bw/day. At 750 mg/kg bw/day they were accompanied by changes in clinical chemistry (higher ALAT and ASAT activities, higher bile acids and bilirubin levels, statistically significant in males) and statistically significantly increased absolute and relative liver weights. Furthermore, coagulative necrosis was observed starting in 4 males at 750 mg/kg bw/day (up to moderate) and at 750 mg/kg bw/day in 2 females (up to slight). Peribiliary fibrosis was observed at 750 mg/kg bw/day in all 5 males (up to moderate) and all 5 females (up to slight). Starting from 250 mg/kg bw/day increased incidence and severity of hematopoiesis in the spleen was observed in males, which correlated with higher spleen weights and haematological changes including lower blood cell counts, haemoglobin and haematocrit and higher reticulocyte counts at 750 mg/kg bw/day. However, these changes were considered non-adverse based on the absence of inflammatory, degenerative or proliferative lesions. Nevertheless, these effects are considered to be relevant for risk assessment purposes. At 750 mg/kg bw/day urothelium hyperplasia was observed at increased incidence and severity in all selected males and females up to slight degree. Although the hyperplasia was in general diffuse and without cellular atypia, based on the proliferative character this finding was regarded as adverse.

Based on the observed effects, the NOAEL for general parental toxicity was set at 75 mg/kg bw/day.

The treatment at the highest dose level of 750 mg/kg bw/day had an adverse effect on the oestrous cycle, precoital time and the total number of pregnant. Two females in the high-dose group were acyclic and two other had a longer precoital time in comparison with controls. Both occurrence of acyclic estrous cycles and a precoital time of 12 days are infrequent occurrences in this type of study.

In addition, two females were not pregnant and one had a total litter loss. These effects are considered to represent an adverse effect on reproduction. The NOAEL for reproductive toxicity was therefore set at 250 mg/kg bw/day.

At 750 mg/kg bw/day, a lower post-implantation survival index was recorded (67% vs. 93% in the control group) together with the lower litter size (6.6 vs. 11.0 in the control group). These represented adverse developmental effects. Furthermore, at 250 mg/kg bw/day 9 pups went missing between PND 1 -4. This incidence was considered higher than normally encountered in this type of study. At 750 mg/kg bw/day, no higher postnatal loss was recorded. However, it is conceivable that at 250 mg/kg bw/day pup death occurred at a later stage during development than at 750 mg/kg bw/day where post-implantation loss (i.e. earlier during development) may have resulted in a lower number of pups (lower litter size and lower total number of litters). Therefore the NOAEL for developmental effects was set at 75 mg/kg bw/day.