Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2015
Deviations:
no
Remarks:
The study integrity was no adversely affected by any deviations.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch 605803
- Expiration date of the lot/batch: 31 December 2016
- Purity test date: 10 October 2016
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article was homogenized in DMSO within 6 hours of dosing.
FORM AS APPLIED IN THE TEST: The test article was homogenized in DMSO within 6 hours of dosing.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl: WI(Han)(outbred, SPF-Quality)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Males: 10-12 weeks, Females: 12-14 weeks
- Weight at study initiation: Males: Mean: 315.4 g; Females: Mean: 222 g
- Fasting period before study: None
- Housing:
Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages,MIV type, height 18 cm) with a maximum of 5 animals/cage.Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups,pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
General: Sterilized sawdust as bedding material (Lignocel S 8-15, JRS -J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg,Germany) and paper as cage-enrichment/nesting material(Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material,food and water.
- Diet: Pelleted roden diet (SM R/M-Z from SSNIFF Spezialdiaten GmbH, Soest, Germany) ad libitum
- Water: Tap water ad libitum
- Acclimation period: At least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 08 June 2016 To: 28 July 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
DMSO
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test article was homogenized in DMSO within 6 hours of dosing.
VEHICLE
- Justification for use and choice of vehicle (if other than water): Test article solubility
- Concentration in vehicle: The dosing solutions were prepared to properly dose 0 (vehicle control), 10, 30, 100, and 300 mg/kg/day test article.
- Amount of vehicle (if gavage): The dose volume was 0.5 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (10 June 2016) according to a validated method (Test Facility Study No. 511828). Samples of formulations were 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 were exposed for 29 days (i.e. for Group 1-4 males during 2 weeks prior to mating, during
mating, and up to termination; Group 5 males were not mated). Group 1-4 females were exposed for
51-63 days, i.e. during 2 weeks prior to mating, during mating, during postcoitum, and during 13-15
days of lactation. Females which failed to deliver healthy offspring were exposed for 17-41 days.
Group 5 (300 mg/kg/day) females were exposed for 29 days. In consultation with the sponsor, it was decided not to pair these females since excessive parental toxicity at this dose level could hamper toxicological interpretation of any reproductive/developmental effects.
Frequency of treatment:
Daily (during treatment periods where appropriate)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control (DMSO), Group 1
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 4
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 5
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a 10-day range finding study.
- Rationale for animal assignment: Random
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstance of any death was recorded in detail.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals, at least after treatment (on the peak period of anticipated effects after treatment). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.
BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. Group 4 and 5 animals were also weighed on Day 13 in order to monitor the health status of the animals.
FOOD CONSUMPTION: 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:
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment-related effects was suspected.
OTHER: Ophthalmoscopic examination was conducted during the last week of treatment for Group 1-5 males and Group 5 females, and during the last week of lactation for Group 1-4 females. Both eyes were examined by means of an ophthalmoscope, following instillation of topicamide solution.
Blood collection: Blood samples were collected at the end of the treatment period on the day of sheduled necropsy for all Group 5 animals and half of the animals in the other treatment groups. Blood samples were collected under anaesthesia using isofluorane. The animals were depreived of food overnight before blood sampling, but water was available. Blood samples were drawn from the retroorbital sinus.
The following hematological parameters were examined:
White blood cells,
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
Red blood cells
Reticulocytes
Red blood cell distribution width
Hemoglobin
Hematocrit
Mean corpuscular volume
Mean corpuscular hemoglobin
Mean corpuscular hemoglubin concentration
Platelets
Prothrombin Time
Activation Partial Thrmboplastin Time
The following clinical chemistry parameters were examined:
Alanine aminotransferase
Aspartate aminotransferase
Alkaline Phosphatase
Total protein
Albumin
Total Bilirubin
Bile acids
Urea
Creatinine
Glucose
Cholesterol
Sodium
Potassium
Chloride
Calcium
Inorganic Phosphate
Sacrifice and pathology:
SACRIFICE
- Male animals: All surviving animals: Following completion of the mating period (after 29 days of dose administration).
- Maternal animals: All surviving animals
Group 5 females: After 30 days of dose administration.
Group 1-4 females which delivered: PND 14-16.
Female no. 70 (10 mg/kg/day, Group 2) with total litter loss: Within 24 hours of litter loss.
Other:
Spontaneous deaths: As soon as possible after death and always within 24 hours.
Euthanized in extremis: When pain, distress or discomfort was considered not transient in nature or was likely to become more severe.

GROSS NECROPSY
After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The number of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites are present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites (Salewski staining prepared at Charles River
Den Bosch using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues from all Group 5 animals and 5/sex from Groups 1-4 were collected:
Adrenal glands (Esophagus)
(Aorta)
Ovaries
Brain - cerebellum, mid-brain, cortex (7-levels)
(Pancreas)
Caecum
Peyer's patches [jejunum, ileum] if detectable
Cervix
Pituitary gland
Clitoral gland
Preputial gland
Colon
Prostate gland
Coagulation gland
Rectum
(Cowper’s gland)
(Salivary glands - mandibular, sublingual)
Duodenum
Sciatic nerve
Epididymides
Seminal vesicles
Eyes (with optic nerve (if detectable) and Harderian gland)
Skeletal muscle
(Skin)
Mammary gland area (males and females)
Spinal cord -cervical, midthoracic, lumbar
Femur including joint
Spleen
(Glans penis)
Sternum with bone marrow
(Levator ani plus bulbocavernosus muscle complex (LABC))
Stomach
Testes
Heart
Thymus
Ileum
Thyroid including parathyroid if detectable
Jejunum
(Tongue)
Kidneys
Trachea
(Lacrimal gland, exorbital)
Urinary bladder
(Larynx)
Uterus
Liver
Vagina

All gross lesions
Lymph nodes - mandibular, mesenteric
Tissues/organs in parentheses were not examined by a pathologist, as no signs of toxicity were noted at macroscopic examination.
From all remaining Group 1-4 animals, males that failed to sire, females which failed to deliver and females with total litter loss, the following organs and tissues were collected and fixed in 10%
buffered formalin:
Cervix
Preputial gland
Clitoral gland
Prostate gland
Coagulation gland
Seminal vesicles
Cowper’s glands
Testes
Epididymides
Thyroid including parathyroid if detectable
Glans penis
Uterus
Levator ani plus bulbocavernosus muscle complex (LABC)
Vagina
All gross lesions
Mammary gland area (males and females)
Ovaries
Organ weights were recorded for the following:
Adrenal glands
Brain
Cowper’s glands
Epididymides
Glans penis
Heart
Kidneys
Levator ani plus bulbocavernosus muscle complex (LABC)
Liver
Ovaries
Prostate
Seminal vesicles including coagulating glands
Spleen
Testes
Thymus
Thyroid (including parathyroid if detectable)
Uterus (including cervix)

Blood sampling for Thyroid Hormone Analysis:
F0-generation, males and females:
End of study from all animals at planned necropsy; this included Group 1-4 females on Day 14-16 of lactation, all females that failed to deliver healthy pups and all males and Group 5 females after at least 4 weeks of treatment (including all males that failed to sire). Blood samples were collected under anaesthesia using isoflurane between 7.00 and 10.30 a.m.. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples (0.9 mL) were drawn from the retroorbital sinus and collected into serum tubes (Greiner Bio-One GmbH, Kremsmünster, Austria). After clotting and centrifugation, serum was used as listed below.
Males: 1 aliquot of 150 μL serum was used for measurement of thyroxine (T4), and the remaining
volume of serum for measurement of thyroid-stimulating hormone (TSH).
Females: The serum was stored for possible measurement of thyroxine (T4) and/or thyroidstimulating hormone (TSH).
Serum samples were stored at ≤-75°C. Under these storage conditions, the samples are stable for 6 months. Any samples remaining after 6 months were discarded.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs that were considered to be related to treatment were observed among surviving animals down to the lowest dose level of 10 mg/kg/day. At 300 mg/kg/day, clinical signs observed among surviving animals primarily consisted of opacity of the eyes which was noted among all animals from Week 2 of treatment onwards. Other clinical signs occurring at lower frequency included hunched posture (1/7 males and 6/6 females), piloerection (1/6 females), rales (2/7 males and 6/6 females), laboured respiration and gasping (1/6 females). At 100 mg/kg/day, clinical signs observed among surviving animals consisted of hunched posture (3/10 males, 1/6 females), piloerection (2/10 males, 3/6 females), rales (6/10 males, 4/6 females) and gasping (1/10 males). At 30 mg/kg/day, clinical signs observed among surviving animals consisted of rales (3/10 males, 1/10 females) and piloerection (6/10 females). At 10 mg/kg/day, clinical signs observed among surviving animals consisted of hunched posture and piloerection in 1/10 females.
The occurrence of scabs and rales among individual control animals and scabs at 30 mg/kg/day 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. Salivation seen after dosing among all animals at 30, 100 and 300 mg/kg/day, and for females also at 10 mg/kg/day and in the control group 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). This sign may be related to irritancy/taste of the test item. No clinical signs additional to those recorded at the daily detailed clinical observations were noted during weekly arena observations.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were eleven premature decedents: At 100 mg/kg/day, 2/10 females were found dead on Days 8 and 27, respectively, and 2/10 females were sacrificed moribund on Days 12 and 17, respectively. At 300 mg/kg/day, 3/10 males and 4/10 females were sacrificed moribund between Days 10 and 27. Main reasons for premature sacrifices consisted of abdominal swelling and respiratory distress (gasping). At 100 mg/kg/day, female no. 86 was sacrificed in extremis on Day 17 with forestomach erosion/ulceration as main cause for moribundity. This female showed body weight loss (11%) over Weeks 1 and 2 of treatment. For female 85 sacrificed in extremis on Day 12, the cause for moribundity remained unclear, while for females 82 and 88 this was likely gavage related (marked diffuse ulceration of trachea). Gasping and/or abdominal swelling were noted for all these animals prior to sacrifice/death. At 300 mg/kg/day, a total of seven rats (male nos. 42, 43 and 45 and female nos. 91, 93, 97 and 99) were sacrificed for humane reasons. For three of these animals (male nos. 42 and 45 and female no. 99) forestomach erosion/ulceration was noted which was considered to have contributed to their moribundity. For female 93, sacrificed in extremis on Day 10, the cause for moribundity remained unclear, while for female 97 this was likely gavage related (marked ulceration of trachea and bronchus epithelium). For five animals (nos. 42, 43, 45, 91 and 99), test item-related findings in the cornea of the eye were recorded, consisting of stromal edema and vacuolation of the epithelium. A correlation could be made with cloudy eyes noted at necropsy. At lower frequency, piloerection, hunched posture, bleeding from the nose, gasping, rales, laboured respiration and/or abdominal swelling were noted for these animals.
There were no premature decedents in both sexes of the control group and the 30 mg/kg/day groups and in males of the 100 mg/kg/day group. One female at 10 mg/kg/day (no. 70) was sacrificed due to total litter loss.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg/day, reduced mean body weight gain and mean body weights were recorded throughout treatment for males (statistically significant on all occasions). For females at this dose, slight mean weight loss/lower weight gain was recorded during treatment, which was mainly ascribed to weight loss of female nos. 91 and 98. Mean body weights were also lower for these females during lactation (statistically significant over Days 4-13). At 100 mg/kg/day, mean body weight gain / body weight of males was lower than controls, being statistically significant on Day 8 of the premating period and/or on Day 1 of the mating period (Day 15 of treatment). For females at this dose, slight mean body weight loss was noted during the first week of treatment which was mainly attributed to weight loss
for female no. 86. Throughout lactation, mean body weight gain appeared slightly lower than controls (but not statistically significant). At 10 and 30 mg/kg/day, any statistically significant changes in mean body weights occurred in the absence of a dose-related trend, and were not considered to betreatment-related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
At 300 mg/kg/day, a lower food consumption was recorded for males and females during the first week of treatment. On subsequent treatment weeks, no clear differences in food intake were noted, also taking into account mean food intake levels recorded for other dose groups. At 100 mg/kg/day, food consumption was lower for females towards the end of the lactation period and during lactation, being statistically significant on some occasions. At 10 and 30 mg/kg/day, food consumption before or after correction for body weight was similar between treated and control animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
Description (incidence and severity)
At 300 mg/kg/day, buphthalmos was recorded for 5/7 surviving males and 6/6 surviving females, and focal corneal opacity, anterior lens opacity and pinpoint corneal opacities was noted for all surviving animals (7/7 males and 6/6 females). For one surviving female at 300 mg/kg/day (no. 98), corneal ulceration was noted. Structures underlying the cornea could not be examined for all surviving animals due to opacity of the cornea.
At 10, 30 and 100 mg/kg/day, no treatment-related ophthalmology findings were noted, as the nature and incidence of these findings was similar among the groups, and occurred within the range considered normal for rats of this age and strain.
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes occurred in haematological parameters of treated rats. Any statistically significant changes occurring among the dose groups were considered to be of no toxicological relevance since there were no supportive changes in other red blood cell parameters, changes were slight in nature and/or did not show dose-related trend. Haematology parameters of females at 300 mg/kg/day (determined at Week 4 of treatment) were considered unaffected by treatment (compared to control data from non-mated female rats aged approximately 19 weeks). Any statistically significant changes compared to the control group were therefore considered unrelated to treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes in clinical biochemistry parameters distinguished animals from control animals:
• Higher alkaline phosphatase activity (ALP) in males and females at 300 mg/kg/day (for females compared to control data from non-mated female rats aged approximately 19 weeks).
• Higher total bilirubin in males at 300 mg/kg/day.
• Higher urea in females at 100 mg/kg/day.
• Higher cholesterol in females at 100 mg/kg/day (not statistically significant but higher than expected for rats of this age and strain).
• Higher bile acids in males at 300 mg/kg/day and in females at 100 mg/kg/day.
• Higher inorganic phosphate in males at 300 mg/kg/day and in females at 100 mg/kg/day.
The higher mean creatinine in females at 100 mg/kg/day occurred due to a single high value for female no. 81. No such change was noted for other animals at this dose, nor at the next higher dose of 300 mg/kg/day (even though for females at 300 mg/kg/day the dosing period was shorter than at 100 mg/kg/day). Therefore this was not considered to be related to treatment.
The lower aspartate aminotransferase activity (ASAT) in males was considered to be of no toxic ological relevance since an opposite and more pronounced effect would be expected in case of target organ toxicity. Any other statistically significant changes in clinical biochemistry parameters for females at 300 mg/kg/day were considered to be unrelated to treatment since means were within the range expected for non-mated female rats aged approximately 19 weeks. At 10 and 30 mg/kg/day, clinical biochemistry parameters were not considered to be affected by treatment.
Serum levels of T4 in F0 males were not considered to be affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
At 300 mg/kg/day, mean motor activity (total movements) of males at 300 mg/kg/day appeared lower than controls (not statistically significant). For ambulations, there was no apparent treatment-related difference among the male groups. For other dose groups (including for females at 300 mg/kg/day), the variation in motor activity did not indicate a relation with treatment. Motor activity of females (determined in Week 4 of the study instead of at Lactation) was within the range expected for rats of this age and strain. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. Hearing ability, pupillary reflex and static righting reflex were normal in all examined males and females. Grip strength of males (Week 4) and females (Lactation) was similar between the groups. Grip strength of females at 300 mg/kg/day (Week 4) was within the range expected for rats of this age and strain. For females at 300 mg/kg/day, pupillary reflex could not be
assessed due to opacity of the eyes.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher liver weights (relative to body weights) were noted in the 300 mg/kg/day group males. Test item-related higher liver weights (absolute and relative to body weights, when compared to historical control data) were also noted in the 300 mg/kg/day group females. This group offemales had a shorter treatment duration and were not mated. Test item-related lower thymus weights (absolute) were noted in the 100 mg/kg/day group females. The lower absolute and relative weights of some organs in males at 300 mg/kg/day were regarded to be caused by their lower body weight.
There were no other test item-related organ weight changes.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The following test item-related macroscopic findings were observed:
• (Fore) stomach, irregular surface in 3/10 females treated at 100 mg/kg/day (3/4 unscheduled deaths) and in 8/10 males (3/3 unscheduled and 5/7 scheduled) and 9/10 females (4/4 unscheduled, 5/6 scheduled) at 300 mg/kg/day.
• Forestomach wall/limiting ridge thickened in 1/10 males (1/7 scheduled death) and 3/10 females treated at 300 mg/kg/day (3/4 unscheduled deaths).
• Forestomach, several tan foci in 1/10 females (1/6 scheduled death) of the 300 mg/kg/day.
• Eyes, cloudy in 10/10 males (3/3 unscheduled and 7/7 scheduled) and 7/10 females (1/4 unscheduled, 6/6 scheduled) treated at 300 mg/kg/day.
Unscheduled sacrificed rats additionally showed the following macroscopic test item-related findings:
• GI tract, distended with gas in 3/4 females at 100 mg/kg/day and in 3/3 males and 3/4 females at 300 mg/kg/day. The microscopic correlate was flattened cecum epithelium
or luminal dilation of the colon (normal histology but the mucosa was somewhat compressed).
• Adrenal glands, enlarged in 1/4 females at 100 mg/kg/day and 2/3 males and 1/4 females at 300 mg/kg/day. The microscopic correlate was cortical hypertrophy.
• Spleen, reduced in size in 3/4 females at 100 mg/kg/day and in 1/3 males and 1/4 females at 300 mg/kg/day. There was no microscopic correlate.
• Thymus, reduced in size in 3/4 females at 100 mg/kg/day and in 2/3 males and 3/4 females at 300 mg/kg/day. The microscopic correlate was lymphoid atrophy.
The remainder of the recorded macroscopic findings of the scheduled sacrifices was 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 MTDID 15670 were noted in the scheduled sacrificed rats in the stomach, eyes, thymus, bone marrow (sternum and/or femur), adrenal glands, liver and kidneys.
Forestomach:
• Erosion/ulceration was noted in 1/6 males (minimal) and 4/6 females (up to slight) of the 300 mg/kg/ day group.
• Squamous cell hyperplasia was noted in 5/6 males and 5/5 females of the 300 mg/kg/day group (both sexes up to moderate).
• Hyperkeratosis was noted at increased incidence and severity in both sexes of the 300 mg/kg/day group (4/6 and 5/6, up to slight), compared to minimal severity in 1/5 control female, 1/5 females at 10 mg/kg/day and in 2/5 males at 100 mg/kg/day.
• Inflammation (lymphogranulocytic) was noted in a few rats of the 30 and 100 mg/kg/day group (minimal) and in 5/6 males and 2/6 females of the 300 mg/kg/day group (up to slight). In the individual rats of the 30 and 100 mg/kg/day groups, this finding was not accompanied by concomitant lesions of the forestomach (besides a single case of hyperkeratosis). Since low incidences and severities of inflammation can be seen as background finding, this finding at 30 and 100 mg/kg/day was considered as unrelated to treatment. In the 300 mg/kg/day group, the inflammation was accompanied by hyperplasia and/or erosion/ulceration and/or edema and therefore considered to be test item-related.
• Edema was noted in 2/6 males (up to slight) and 1/6 females (minimal) of the 300 mg/kg/day group.
Glandular stomach:
• One or more of the following test item-related findings were present at minimal severity in 1 or 2 males of the 300 mg/kg/day group: Mucosal hypertrophy, increased basophilia of proliferation zone, increased eosinophilic globules and/or mucosal hemorrhage mucosa.
Eyes:
• Cornea stromal edema, (bilateral) was noted in 5/5 males and 1/5 females of the 100 mg/kg/day group (minimal) and in 7/7 males and 6/6 females of the 300 mg/kg/day group (up to moderate).
• Cornea epithelium vacuolation was noted in 1/5 males of the 100 mg/kg/day group (minimal) and in 7/7 males and 6/6 females of the 300 mg/kg/day group (up to moderate).
Thymus:
• Lymphoid atrophy (minimal) at increased incidence was present in 4/5 females of the 100 mg/kg/day group and in 3/5 males and 2/6 females of the 300 mg/kg/day group, compared to in 1/5 females of the 30 mg/kg/day group.
• Increased lymphocytolysis was observed in 1/5 females of the 100 mg/kg/day group (minimal).
Bone marrow, sternum and femur:
• Inflammation, mixed or granulomatous mainly present in the femur was noted in 1/5 females of the 30 mg/kg/day group (slight) and in 3/5 females of the 100 mg/kg/day group (up to moderate). Large macrophages and multiple small areas with nuclear debris were the most prominent features of the inflammation and therefore the modifier granulomatous was used.
• Subtle changes in the bone marrow cellularity of the sternum or femur, recorded as fatty replacement and/or increased myelopoiesis or decreased erythropoiesis were noted in 2/5 females of the 100 mg/kg/day group (up to slight) and in males and females of the 300 mg/kg/day group (minimal).
Adrenal glands:
• Vacuolation zona fasciculata at increased incidence was noted in 5/5 males of the 300 mg/kg/day group (minimal).
Liver:
• Minor increased incidence of hepatocellular hypertrophy in two males of the 300 mg/kg/day group (minimal) related with higher liver weight (relative to bodyweight) compared to single incidences at 0 and 100 mg/kg/day.
Kidneys:
• Tubular vacuolation in the medulla was noted in 4/5 of the 300 mg/kg/day group (up to slight). Test item-related microscopic findings of the unscheduled sacrificed rats were similar to the scheduled sacrificed rats. They were, in general, present in both sexes (except for trachea and lung findings that were only observed in females) of the unscheduled sacrifices and in general present at higher severity in unscheduled sacrifices compared to the scheduled sacrifices.
These findings were observed in:
• Forestomach, erosion/ulceration (up to marked), hyperplasia/hyperkeratosis (up to moderate), inflammation (up to slight), edema (slight).
• Glandular mucosa of the stomach, mucosa hypertrophy (minimal), hemorrhages (minimal).
• Eyes, cornea (bilateral) stromal edema (up to moderate), cornea vacuolation epithelium (up to moderate). Bone marrow, fatty replacement (up to slight), increased myelopoiesis (up to moderate), and decreased erythropoiesis (up to slight).
• Thymus, lymphoid atrophy up to marked and increased lymphocytolysis at slight degree).
• Adrenal glands, vacuolation zona fasciculata (up to slight).
• Kidneys, tubular vacuolation (up to slight).
• Trachea and/or lung, ulceration (marked), considered to be gavage related (therefore, histopathological examination was not extended to intermediate dose groups).
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.
Spermatogenic profiles of all males examined were normal.
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 10 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
mortality
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
bone marrow
Treatment related:
yes
Dose response relationship:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
eye
Organ:
cornea
Treatment related:
yes
Dose response relationship:
yes

Applicant's summary and conclusion

Conclusions:
Based on the results of the study, the parental No Observed Adverse Effect Level (NOAEL) of the test article is 10 mg/kg/day, the reproductive NOAEL is 30 mg/kg/day and the developmental NOAEL is 100 mg/kg/day.
Executive summary:

The repeat dose and reproductive and developmental toxicity of the test article was evaluated in male and female Wistar Han rats. The study was conducted under OECD GLP (1997) conditions. The study method was based on OECD Guideline 422 (2015). SPF-bred Wistar Han rats (10/sex/group) received 0 (100% dehydrated dimethyl sulfoxide vehicle), 10, 30, 100, or 300 mg/kg/day MTDID 15670 via daily oral gavage at a dose volume of 0.5 mL/kg. Males were exposed for 29 days (i.e. for Group 1-4 males during 2 weeks prior to mating, during mating, and up to termination; Group 5 males were not mated). Group 1-4 females were exposed for 51-63 days, i.e. during 2 weeks prior to mating, during mating, during postcoitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were exposed for 17-41 days. Group 5 females were exposed for 30 days. In consultation with the Sponsor it was decided not to pair these females since excessive parental toxicity at this dose level could hamper toxicological interpretation of any reproductive/developmental effects.The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment, and PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: mating, fertility and conception indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy). Formulations were analyzed once during the study to assess accuracy and homogeneity.

Parental animals: At 100 and 300 mg/kg/day, treatment-related mortality occurred. At 100 mg/kg/day, 4/10 females were found dead or sacrificed moribund. At 300 mg/kg/day, 3/10 males and 4/10 females were sacrificed moribund. For most of these animals the cause of moribundity/death established histopathologically was a marked ulceration of the trachea and/or lungs or forestomach erosion/ulceration. Main reasons for their premature sacrifice/death consisted of abdominal swelling and respiratory distress (gasping). This abdominal swelling was considered to reflect distension of the gastro-intestinal tract with gas noted for some animals at necropsy, which correlated microscopically to flattened cecum epithelium or dilated colon lumen.

Forestomach erosion/ulceration was also present in scheduled sacrificed males and females of the 300 mg/kg/day group. In general, most stomach lesions were present in the forestomach of both sexes and also consisted of hyperplasia and/or hyperkeratosis, increased incidence of inflammation, and/or (sub) mucosal edema. Their combined occurrence with erosion/ulceration at these dose levels was considered to represent an adverse effect.

In surviving males of the 300 mg/kg/day group, the glandular mucosa of the stomach was also affected by low incidence and severities of mucosal hypertrophy, increased basophilia of the proliferation zone, increased eosinophilic globules and/or mucosal hemorrhage. These latter findings may reflect a non-adverse local response to direct exposure by oral gavage with the test item. Overall the forestomach and glandular stomach effects are indicative of irritating potential of the test item, affecting the protective epithelial layers.

Trachea and/or lung ulceration observed for three unscheduled deaths were also considered to have been caused through direct contact with the test item, most likely due to aspiration and/or as a resultant of the gavage procedure.

Opacity of the eyes noted for all animals, including most premature sacrifices, at 300 mg/kg/day from Week 2 of treatment onwards. This was accompanied by buphthalmos diagnosed at ophthalmoscopic examination for all animals, along with opacity of the cornea and lens, and corneal ulceration for one surviving female. Correlating microscopy consisted of bilateral cornea stromal edema and vacuolation of cornea epithelial layer (the exterior layer of the cornea) both up to moderate degrees. The transparency of the cornea is to some degree due to its relatively dehydrated normal state (compared with other tissues) and to the orderly stromal lamellar architecture. Increased stromal fluid (edema) increases corneal hydration and disrupts the lamellar arrays, thus resulting in loss of transparency. Therefore, these rats were considered to have had serious vision problems and as such these findings were considered to be adverse. The intra-ocular components of the eye, like lens and retina, did not show test item-related findings. Therefore, it was considered likely that the cornea was affected by a direct repeated exposure of the eye to the test-item, possibly during grooming.

An adverse granulomatous inflammation of the sternal/femoral bone marrow was noted in one surviving female at 30 mg/kg/day and three surviving females at 100 mg/kg/day. This finding was absent in females of the 300 mg/kg/day group (which had a shorter treatment period and were not mated). The inflammation was multifocal and the inflammatory cells were mixed (mainly granulocytes and macrophages) at lower severity and granulomatous (large macrophages) at moderate degree. In three rats the inflammation was observed in the bone marrow of the femur and in one 100 mg/kg/day female also in the sternal bone marrow. These four females were all scheduled sacrifices and without test item-related findings in other organs. Since inflammation of the bone marrow is a very rare lesion and inflammation with cellular debris can be regarded as a degenerative finding, the bone marrow inflammation was considered to be adverse.

A lower motor activity (total movements) was recorded for males at 300 mg/kg/day, which occurred in the absence of concurrent changes in other functional observations tests at this dose. It is conceivable that the lower motor activity occurred secondary to the reduced health status of these animals. At 100 and 300 mg/kg/day, this reduced health status was expressed by clinical signs consisting of hunched posture, piloerection, rales, labored respiration and gasping, next to abdominal swelling and respiratory distress discussed above. Also, lower body weights/slight weight loss was observed at 100 and 300 mg/kg/day for males and females, which for males was already apparent during the initial weeks of treatment, while for females at 100 mg/kg/day this became apparent essentially during lactation. At 30 mg/kg/day, clinical signs among surviving animals were confined to rales and piloerection, whereas at 10 mg/kg/day these consisted of hunched posture and piloerection in a single female. Given the incidental nature of these clinical signs at 10 and 30 mg/kg/day, absence of any concurrent changes in eg. bodyweight and food intake or treatment-related morphological changes, these were considered not toxicologically relevant at these dose levels.

Changes in clinical biochemistry parameters consisted of higher alkaline phosphatase activity

in males and females at 300 mg/kg/day, higher total bilirubin in males at 300 mg/kg/day, higher urea and cholesterol in females at 100 mg/kg/day, higher bile acids and higher inorganic phosphate in males at 300 mg/kg/day and in females at 100 mg/kg/day. These changes were generally mild in nature and had no histopathological correlates. As such, although these changes may be related to treatment, they were considered not adverse.

Non-adverse histopathological changes in scheduled and/or unscheduled sacrifices were noted in the sternal/femoral bone marrow (shift in bone marrow cells), thymus and spleen which were most likely stress-related. A shift in bone marrow cells was recorded for females at 100 mg/kg/day and in males and females at 300 mg/kg/day, generally at a minimal or slight degree. In most rats, fat replaced the early stages of bone marrow cells, sometimes the late stages of primarily granulocytes and sometimes the ME-ratio (myeloid/erythroid ratio) was increased with or without fatty replacement (recorded as increased myelopoiesis and decreased erythropoiesis). Since the majority of the affected rats showed a shift, rather than atrophy, and since in some rats, there appeared to be a relation with forestomach ulceration/inflammation at 300 mg/kg/day, these findings were not considered as stress-related findings. Adversity of this bone marrow cell shift should be evaluated within context of concomitant findings (i.e. forestomach ulceration and moribundity). Therefore at 300 mg/kg/day, the shift in bone marrow cells related with concomitant forestomach ulceration/erosion/inflammation was considered adverse in unscheduled sacrificed males and scheduled and unscheduled sacrificed females.

Non-adverse increased incidence of lymphoid atrophy of the thymus in females at 100 mg/kg/day and in rats of both sexes at 300 mg/kg/day correlated with lower thymus weights and reduced thymus size in females at 100 mg/kg/day. In combination with the statistically significant lower final body weights in both sexes at 100 and 300 mg/kg/day and the high moribundity at 100 and 300 mg/kg/day, the thymus atrophy can be regarded as a stress-related finding. Also the reduced spleen size, without microscopic correlate, observed in unscheduled sacrifices from 100 mg/kg/day onward was regarded to be a stress-related finding.

Other non-adverse histopathological lesions were observed at 100 mg/kg/day and at 300 mg/kg/day. At 100 mg/kg/day these findings consisted of luminal dilation of the colon of males and flattened epithelium of the cecum of females. At 300 mg/kg/day these findings consisted of vacuolation of the tubules in the medulla in kidneys of females, vacuolation of the zona fasciculata of the adrenal glands in males, a minor increase in incidence of minimal hepatocellular hypertrophy in the liver of males, luminal dilation in the colon of both sexes and flattened epithelium of the cecum of both sexes. Those findings were present at low severity and were not accompanied by inflammation or proliferative lesions and therefore considered as non-adverse.

There were no morphological findings in the reproductive organs of either sex after treatment up to 100 mg/kg/day which could be attributed to the test item. Spermatogenic profiles were normal for all males examined.

No treatment-related changes were noted in haematological parameters up to 300 mg/kg/day.

 

Reproductive results: At 300 mg/kg/day, 6/10 females had an extended di-estrus with or without an irregular cycle. Treatment at this dose was terminated on Day 31 (these animals were not paired). At 100 mg/kg/day, a lower fertility and conception index was recorded; a total of 3 out of 8 females were not pregnant. These treatment-related changes were considered to represent an adverse effect on reproductive performance.

No treatment-related changes were noted in any of the other reproductive parameters investigated in this study (i.e. mating index, precoital time, and number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

 

Developmental results: No toxicologically relevant changes in developmental parameters were observed up to the highest dose level at which pups were delivered (100 mg/kg/day).

A lower mean pup body weight was recorded for both sexes at 100 mg/kg/day on Day 13 of lactation, which was approximately 9% lower than controls. In absence of any other treatment-related changes in developmental parameters at this dose, this change was considered not adverse. Also, it is conceivable that these lower pup body weights occurred secondarily to the lower food intake and body weights of females during lactation, and did therefore not represent a primary developmental effect.

No treatment-related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, anogenital distance (PND 1), areola/nipple retention (PND 13 males), T4 thyroid hormone levels (PND 13-15) and macroscopy.

 

Based on the results of the study, the parental No Observed Adverse Effect Level (NOAEL) of the test article is 10 mg/kg/day, the reproductive NOAEL is 30 mg/kg/day and the developmental NOAEL is 100 mg/kg/day.