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Repeated dose toxicity: oral

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

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:
22 March 1996
Deviations:
yes
Remarks:
Several deviations from the study protocol occurred but are not expected to influence the overall test results. Deviations included minor dosing errors and errors with data collection/test performance.
GLP compliance:
yes (incl. certificate)
Remarks:
Bayrisches Landesamt für Gesundheit und Lebensmittelsicherheit
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 10-11 wks
- Weight at study initiation: (P) Males: 241 - 276 g; Females: 167 - 193 g
- Housing: 2 animals / sex / cage in IVC cages (type III H, polysulphone cages) during the premating
period for both males and females and during postmating period for males depending on the mating
status. During mating period males and females were housed together in ratio 1:1 (male to female
). Animals of the recovery groups were housed in groups of 2 animals / sex / cage. In each cage Alt
romin saw fibre was used as bedding.
- Diet: Free access to Altromin 1324 maintenance diet access to Altromin 1324 maintenance diet
- Water: Free access to tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hr light/12 hr dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item formulation was prepared with corn oil. The test item was weighed on a suitable precision balance into a plastic vial which was flooded with argon and the vehicle was added to give the appropriate final concentration of the test item. Formulations were vortexed for 2-3 minutes. The test item formulation was prepared at least once every ten days based on available stability data. Formulates were kept under magnetic stirring during the daily admin
istration.

VEHICLE
- Concentration in vehicle: undiluted, ≤ 0.1 % of water content
- Amount of vehicle (if gavage): 2 mL/kg body weight
- Lot/batch no. (if required): MKBQ9948V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mean recoveries observed for the low-dose group was between 96.4% and 100.8% of the nominal value, between 91.6% and 98.1% for the mid-dose group and between 92.5% and 100.0% of the nominal value for HD group. The mean recoveries observed in the low-, mid-, and high-dose groups were 98.2%, 95.8%, and 95.4% of the nominal concentration, respectively.

Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%. However one sample (no. 16, mid dose week 3, middle sampling location) did not meet this criterion with a recovery of 89.7%. This was not considered to have relevant impact on the study results as the recovery only narrowly missed defined acceptance criterion and as mean recovery of mid dose week 3 was within the range of 10%.

The coefficients of variation (COV) of the different sampling locations (top, middle, bottom) was between 0.7% and 4.5% in LD dose group, between 0.3% and 1.8% in MD dose group and between 1.1% and 2.6% in HD dose group. All samples were homogenous, as COV was below or equal 10%.
Duration of treatment / exposure:
The animals of the main groups were treated with the test item formulation or vehicle on 7 days per week for a period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
The 4 groups consisted of 10 male and 10 female Wistar rats and additional 6 male and 6 female Wistar rats in the control and high dose recovery group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on the results of a previous dose range finding
study and in consultation with the sponsor.
- Rationale for animal assignment: Randomisation was performed with IDBS Workbook 9.4.0
software.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals of the main groups and the recovery groups. Clinical observations included check for spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling,
as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or
bizarre behaviour were recorded if present.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter, as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), as well as on day 4 post-partum along with the pups. Any
animals prematurely sacrificed were weighed prior to the sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Evaluation conducted during functional observactions
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 5 randomly selected males and females (only lactating females were evaluated) from each main group at the end of the treatment and in all males and females at the end of the recovery period prior to or as part of the sacrifice of the animals
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- Parameters checked in tables 1 and 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 5 randomly selected males and females (only lactating females were evaluated) from each main group at the end of the treatment and in all males and females at the end of the recovery period prior to or as part of the sacrifice of the animals
- Animals fasted: Not specified
- Parameters checked in table 3 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: 5 randomly selected males and females (only lactating females were evaluated) from each main group at the end of the treatment and in all males and females at the end of the recovery period prior to or as part of the sacrifice of the animals
- Metabolism cages used for collection of urine: Not specified
- Animals fasted:Not specified
- Parameters checked in table 4 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the lactation period in 5 randomly selected females (only lactating females were evaluated) outside the home cage using a functional observational battery of tests
- Dose groups that were examined: all dose groups
- Battery of functions tested: Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

IMMUNOLOGY: No

OTHER: Based on treatment-related changes observed in the kidneys of the high-dose group, immunohistochemistry of α2µ-globulin was performed on kidneys of the randomly selected 5 males of all main groups and of males of the recovery groups. Immunohistochemistry was performed with Rat α2µ-Globulin Antibody. The image analysis was performed by the CELL Analysis System. Pictures of kidney slides were taken by an Olympus UC30 camera at a magnification of x10. The positive area on the total area was measured in mm2 and calculated as % on the total area. The relative values of positive structures (α2µ-globulin) were used for descriptive statistics (mean, standard deviation, minimum, maximum).
Sacrifice and pathology:
SACRIFICE
- Male animals: Males were sacrificed after completion of the mating period on treatment days 29 or 30.
- Maternal animals: Females along with their pups were sacrificed on post-natal day 4. Non-pregnant females of the main groups were sacrificed on study day 26 using the sperm-positive vaginal smear.

GROSS NECROPSY
- All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 7 were prepared for microscopic examination and weighed, respectively.
Statistics:
A statistical assessment of the results of body weight, food consumption and litter data was perform ed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Statistical comparisons of data acquired during the recovery period were performed with a Student’s t-Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry were statistically analyzed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics will be performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Moving the bedding was observed transiently in all high-dose males and females, including the high-dose recovery group, during the treatment period of this study. Furthermore, salivation was noted transiently in 8/10 males and 8/10 females of the high-dose group, 1/6 males and 4/6 females of the high-dose recovery group and in 1/10 males on one single day of the low-dose group. Moving the bedding and salivation were observed in short timely relation to dose application and thus were considered to be a sign of discomfort or a local reaction to the test item. These slight clinical signs were not considered as adverse systemic effects. They were not observed during the recovery period of this study.

Low incidences of slight clinical signs like local alopecia, crust or scratch in few animals of the control and/or dose groups were seen without dose dependency and were considered as incidental in nature.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two females of the HD group died prematurely during the course of treatment. On high-dose female was found dead on post-natal day 2 and the other was euthanised in a moribund condition for animal welfare reasons on post-natal day 2.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A treatment-related decrease in male body weight was observed at the high dose. During the mating/postmating period, high-dose males showed a slight, statistically significant loss of body weight (mean body weight change -0.40 g in week 1 and -0.60 g in week 2 compared to +96.0 g and +10.50 g in the control group). Considering the whole treatment period, there was a biologically and statistically significantly lower body weight gain in high-dose males when compared to controls (52% below controls). Thus, mean body weight of high-dose males was slightly but not statistically significantly lower at the end of the treatment period (7% below controls).

In females, transiently and slightly but statistically significantly lower body weight gain was noted in the 2nd week of gestation in the high-dose group when compared to the control group (23% below controls).

In the high-dose male recovery group, body weight development was similarly affected when compared to the main high-dose male group. From the second week of treatment onwards, body weight gain decreased with a statistical significant difference to controls of the recovery group. Body weight gain was 6% below controls in the 2nd week of treatment and 91% below controls in the 3rd week of treatment. In the last week of treatment a slight mean loss of 1.50 g body weight occurred when compared to a gain of 9.00 g in controls. Thus, mean body weight was slightly below controls at the end of the treatment period (7% below controls without statistical significance) and the beginning of the recovery period (8% below controls with statistical significance). After ceasing of treatment with the test item, body weight development was noted to recover in the high-dose male recovery group and showed no statistical or biological difference to controls in the first week of the recovery period. In the 2nd week of the recovery period body weight gain of the high-dose male recovery group was statistically significantly 119% above controls. In the high-dose female recovery group, body weight gain was transiently slightly but statistically
significantly higher in the 4th week of the treatment period. This was followed by a slight but statistically significant loss of mean body weight (- 2.50 g) when compared to a gain of 6.83 g in controls in the last week of treatment. These slight, transient and inconsistent effects were not considered to be in a toxicologically relevant range. During recovery period, body weight development was comparable between the dose group and controls. No effects of toxicological relevance on body weight development were noted in the male and female low-dose and mid-dose group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In males and females, mean food consumption was comparable between the dose groups and the respective controls during the premating period of this study. There were no statistically or biologically significant differences. Furthermore, there was no effect of toxicological relevance on food consumption during gestation period of females. However, food consumption was biologically and statistically significantly reduced in high-dose females during lactation period. Food consumption was 43% below controls. There was no effect in low- and mid-dose females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ophthalmoscopic findings in any of the animals of this study.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Slightly but statistically significantly higher percentage of monocytes was noted in the high-dose males (5.66%) and females (3.96%) when compared to the respective control group (males: 1.54%, females: 2.08%). However, as values were within the normal range of biological variation, this isolated change with only slight differences to controls was not considered to be toxicologically relevant. Prothrombin time (PT) was noted to be marginally but statistically significantly higher in mid-dose males when compared to the control group (9% above controls). As values were within the normal range of variation, this marginal not dose dependent difference was considered as incidental in nature.

At the end of the recovery period, a statistically significant effect was noted on mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) with marginally lower values in the high-dose males recovery group (6% and 8% below controls, respectively) and the high-dose female recovery group (both 6% below controls). No such effect was noted at the end of the treatment period. Furthermore, red blood cell count and hemoglobin was comparable between the high-dose male and female recovery group and the respective controls. Differences to controls were marginal and did not adversely affect the health condition of the animals. There were no clinical signs of anemia such as pale skin or reduced spontaneous activity. Furthermore, there were no microscopic findings in the bone marrow at the end of the recovery period. Thus, these marginal and isolated changes of few red blood cell parameters were not considered as toxicologically relevant.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant but inconsistent changes were observed for specific enzymes in the blood. In males, alkaline phosphatase (AP) was statistically significantly, 58% below controls in the mid-dose group. For high-dose males, mean value was 44% below controls without achieving statistical significance. In females, AP was statistically significantly, 56% below controls in the low-dose group and 62% below controls in the high-dose group. Mean value was 34% below controls in the mid-dose female group without achieving statistical significance. Without dose dependency, differences to controls were considered as incidental and not related to the treatment with the test item. Furthermore, slightly and statistically significantly lower aspartate-aminotransferase (ASAT) in mid- (44% below controls) and high-dose (39% below controls) males also did not follow a dose dependent pattern and was not considered as toxicologically relevant with values in the normal range of variation. No statistically significant effect for ASAT was noted in females.

Slightly but statistically significantly lower mean value for sodium was noted in mid-dose males (15% below controls) but not in females. As this did not follow a dose dependent pattern this isolated change was not considered as toxicologically relevant. There were no statistically significant changes in clinical biochemistry findings during the recovery period for any treatment level.
Urinalysis findings:
no effects observed
Description (incidence and severity):
All parameters of urinalysis of the dose groups at the end of the treatment and the recovery period were not considerably different to the corresponding control group and were within the normal range of variation.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
At the end of the treatment and the recovery period in males and females, no toxicologically relevant effects were observed in any of the tested parameters of the functional observation battery including gait, strength and reflexes.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period, a toxicologically relevant effect was noted for testes weight in the high-dose group. Mean weight of testes was moderately and statistically significantly reduced in high-dose males (absolute weight 26% below controls, relative (to body weight) weight 20% below controls). Furthermore, mean absolute weight of prostate with seminal vesicles and coagulating glands was statistically significantly lower in the high-dose group when compared to the control group (16% below controls).

Slightly and statistically significantly higher mean absolute and relative weight of liver was noticed in high-dose males (absolute weight 23% above controls) and high-dose females (absolute weight 23% above controls).

Mean absolute spleen weight was marginally but statistically significantly lower in mid- (15% below controls) and high-dose (16% below controls) males when compared to the control group. A marginal tendency towards lower spleen weight was also observed in high-dose females with a mean value 19% below controls but which did not achieve statistical significance. At histopathological examination, there were no findings in spleen except for lymphoid atrophy in two high-dose females. Thus, marginal changes of spleen weight observed in high-dose females were not considered as toxicologically relevant due to missing microscopic correlate.

Marginally but statistically significantly lower mean absolute brain weight was found only in high-dose males (5% below controls). This marginal difference to controls was not assumed to be toxicologically relevant as there was no effect in females or in recovery animals and no microscopic correlate.

Pituitary gland also showed a slight tendency towards dose dependently lower weight in the male dose groups when compared to the control group. Absolute weight was 20%, 28% and 32% below controls in males at all three dose levels, respectively, but without statistical significance. Absolute weight was 6%, 14% and 34% below controls in females at all three dose levels, respectively, which achieving statistical significance in high-dose females. However, values showed high inter-individual variability. As there were no statistically or biologically significant differences between the groups at the end of the recovery period, slight weight changes at the end of the treatment period were not considered as adverse systemic effect.

Slightly but statistically significantly lower mean absolute but not relative heart weight was seen in high-dose males (14% below controls) whereas slightly but statistically significantly higher relative (to body weight) but not absolute heart weight was noted in high-dose females (13% above controls). These slight and inconsistent differences to controls without microscopic correlate were assumed to be incidental.

At the end of the recovery period, mean absolute and relative testes weight of high-dose males from the recovery group was slightly and statistically significantly below controls (absolute weight 15% and relative weight 11% below controls).

Slightly higher liver weight was still noticed at the end of the recovery period in males and females. Mean relative (to body weight) but not absolute liver weight was statistically significantly, 18% above controls in the high-dose males from the recovery group. In the high-dose female recovery group, mean absolute and relative (to body weight) weight of liver was statistically significantly, 11% above controls. As microscopic findings in the liver at the end of the treatment period recovered after the treatment-free period, slight changes to liver weight at the end of the recovery period were not considered as toxicologically relevant.

Slightly but statistically significantly higher relative (to body weight) but not absolute mean weight of kidneys was found in the high-dose male recovery group (23% above controls) and the high-dose female recovery group (8% above controls). Without microscopic findings in the kidneys at the end of the recovery period, observed slight changes to kidney weight were not considered as toxicologically relevant.

Relative (to body weight) but not absolute heart weight of the high-dose male recovery group was noted to be slightly but statistically significantly above the respective controls (16% above controls). However, this was not noted in males at the end of the treatment period or in female animals. As there were also no related microscopic finding in the heart examined at the end of the treatment period, this was not assumed to be toxicologically relevant.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Few gross pathological changes were recorded at necropsy in the surviving animals at the end of the treatment period without following a dose dependent pattern.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The testes of all high-dose males were affected by tubular degeneration. Mainly elongated and round spermatids of stages V-VII were affected. More mature spermatids and spermia appeared to be unaffected. In some tubules, there were reabsorbed apoptotic bodies or Sertoli cell vacuolation. In the epididymides, only a minimal severity of cellular detritus (shedded epitehlia and/or pyknotic sperm cells) was recorded in most high-dose males. After the treatment-free recovery period, the findings in testes did not resolve.

In most mid- and high-dose animals, there was a minor severity of hepatocellular hypertrophy in the liver. In one decedent female, there was in addition a multifocal moderate necrosis that may be considered to be of agonal nature. No further hepatic lesion was recorded. Findings resolved after the treatment-free period. In male kidneys, the severity of tubular hyaline inclusions increased at the high-dose group. After evaluation by immunohistochemistry, there were no meaningful differences in the contents of α2-microglobulin between controls and test item-treated groups. In both decedent females, there was tubular cell necrosis. This finding affected mainly the distal tubules. No findings were noted after the treatment-free recovery period.

In one surviving high-dose female, there was an ulceration associated with squamous hyperplasia and inflammation in the forestomach. In another female, there was also forestomach squamous hyperplasia. Ulceration in the glandular stomach and squamous hyperplasia at the limiting ridge of the forestomach were deemed to be stress-related. These findings were associated with diffuse hypertrophy of the zona fasciculata in the adrenal cortex of two high-dose females, lymphoid atrophy of the spleen and lymph node, and bone marrow atrophy in high-dose females, and an increased atrophy of the thymus in both sexes of the high-dose group. No such differences were noted after the treatment-free recovery period.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity

open allclose all
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
A combined repeated dose toxicity study with a reproductive/developmental toxicity screening test was performed with the test substance according to OECD TG 422 and GLP at dose levels of 100, 300 and 1000 mg/kg bw/day. The NOAEL for general systemic toxicity can be established at 300 mg/kg bw/day. General systemic toxicity was based on increased mortality, adversely reduced body weight, food consumption, and histopathology effects to the kidney and testes at the highest dose level.