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Toxicological information

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
Study period:
21 Jan - 30 Mar 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Jan - 30 Mar 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 14-15 weeks old
- Weight at study initiation: males: 337 - 395 g; females: 189 - 233 g
- Housing: housed in groups of 5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages during the premating period for both males and females and during post-mating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female).
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The vehicle corn oil was dried and de-acidified. The test item was weighed into a tared glass vial on a suitable precision balance and the vehicle was added (w/v) to give the appropriate final concentration of the test item. The formulation was alternately vortexed and/or stirred until visual homogeneity was achieved. After homogenization the formulation was overlaid with argon to prevent instability caused by repeated contact of the test item formulation with air.
Based on the results of stability testing, the test item formulations were prepared at least every 11 days. The prepared formulation was stored protected from light and at room temperature.
Formulates were kept under magnetic stirring during the daily administration.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected based on the test item’s characteristics and testing guideline.
- Concentration in vehicle: 15.63, 62.5 and 187.5 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg body weight
- Lot/batch no.: MKCH1635, MKCH6411, 2IC0148
Details on mating procedure:
- M/F ratio per cage: Mating was performed using a ratio of 1:1 (male to female).
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage.
Analytical verification of doses or concentrations:
yes
Remarks:
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10 %.
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle as part of a separate GLP study.
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
As the test item was shown to be homogenous (after at least 30 min without stirring), samples were not collected during the study for the investigation of homogeneity and samples were only taken for substance concentration verification in study week 1 (pre-mating period), week 3 (first week of mating), week 5 (gestation) and in the last week of the study (gestation / lactation) from all groups (16 samples).
Concentration analysis: The recoveries observed for the low-dose group was between 93.7% and 99.7% of the nominal value, between 95.6% and 98.2% for the mid-dose dose group and between 94.3% and 99.8% of the nominal value for high-dose group. The mean recoveries observed in the low-, medium- and high-dose groups were 96.9%, 96.6%, and 97.3% of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%.

Duration of treatment / exposure:
Maximum period of 63 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 (PND) 12 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, 7 days/week
Details on study schedule:
Not applicable
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
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. In the dose-range finding study, 3 males and 3 females per group were treated orally with the test item at doses of 100, 300 and 1000 mg/kg bw/day for 14 days. Mortality was observed in 1/3 male animals treated with 1000 mg/kg bw/day. Additionally, main test item-related findings at 1000 mg/kg bw/day were macroscopic findings at the kidney/ureter (enlarged/dilated), a tendency for increased mean kidney weight and increased clinical biochemical kidney parameters (Crea, Urea, TP, Alb). No adverse findings were seen in the dose groups treated with 100 and 300 mg/kg bw/day.
Based on this, the highest dose in this study was set as 750 mg/kg bw/day. The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

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 outside the home cage in a standard arena. Inadvertently, the detailed clinical observation was not performed in week 4 for the five randomly selected female animals.
- Clinical observations included: 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.

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 GD 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4, PND 9 and PND 13 along with pups. All animals were weighed directly before termination. Any animals prematurely sacrificed were weighed prior to their sacrifice.

FOOD CONSUMPTION: Yes
- Food consumption: Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the last week of treatment in males and during the last week of lactation in females
- Dose groups that were examined: all dose groups (5 randomly selected animals)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the time of sacrifice
- Anaesthetic used for blood collection: Yes; ketamine/xylazin
- Animals fasted: Not specified
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the time of sacrifice
- Animals fasted: Not specified
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each group
- Parameters checked in table 2 were examined.

PLASMA/SERUM HORMONES: Yes
- Time of blood sample collection: at the time of sacrifice
- Animals fasted: Not specified
- How many animals: adult males

URINALYSIS: Yes
- Time schedule for collection of urine: at the time of sacrifice
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the last week of the lactation period in 5 randomly selected females. Inadvertently, functional observations of the 5 randomly selected animals were not performed before treatment for female animals, as well as for male animals of the low-, mid- and high-dose groups.
- Dose groups that were examined: all dose groups
- Sensory reactivity to different modalities was assessed: 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: No
Oestrous cyclicity (parental animals):
Oestrous cycles were monitored before treatment started to select for the study females with regular oestrous cyclicity. Vaginal smears were examined daily from the beginning of the treatment period until evidence of mating.
Vaginal smears were also examined on the day of necropsy to determine the stage of oestrous cycle.
Sperm parameters (parental animals):
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring: number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Additionally, any abnormal behavior of the offspring was recorded.
The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD/Cube root of pup weight). The number of nipples/areolae in male pups was counted on PND 12.

Bood samples from 2 pups/litter (1 male and 1 female, if possible) at termination on PND 13 were assessed for serum levels for thyroid hormones. Assessment of T4 in blood samples from day 4 pups was not carried out.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving males were sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days) by using anaesthesia (ketamine/xylazin).
- Maternal animals: Females were sacrificed on the respective PND 13 by using anaesthesia (ketamine/xylazin).

GROSS PATHOLOGY: Yes
Gross necropsy consisted of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4% neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70% ethanol.

ORGAN WEIGHTS: Yes
- The wet weight of the organs (see Table 4) of 5 sacrificed males and 5 females (only lactating females were evaluated) randomly selected from each main group, as well as of all males and females of the recovery groups was recorded as soon as possible. In addition reproductive organs (testes, epididymides, prostate with seminal vesicles and coagulating glands, uterus with cervix and ovaries) of all animals were weighed.
Thyroid/parathyroid glands from 1 pup/sex/litter/group (if possible) (sacrificed on PND 13) and from all adult males and females were preserved. Weight of thyroid/ parathyroid glands was measured after fixation.

HISTOPATHOLOGY: Yes
The tissues indicated in Table 5 from five randomly selected males and females were preserved in 4% neutral-buffered formaldehyde except for eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol.
A full histopathology was carried out on the preserved organs and tissues (Table 5) of the selected animals of the control and high dose group which were sacrificed at the end of the treatment period. A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study or which were euthanised due to morbidity.
For organs and tissues showing treatment-related changes in the high dose group, these examinations were extended to animals of all other dosage groups. Only organs and tissues of the other dosage groups showing changes in the high dose group were examined.
Any gross lesion macroscopically identified was examined microscopically in all animals. Possible discoloration due to the test item was evaluated in the organs of all dose groups.

Postmortem examinations (offspring):
SACRIFICE
All surviving pups were killed by cervical dislocation on PND 13.

GROSS NECROPSY
- Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
Statistics:
A statistical analysis of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry were statistically analysed 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.
Reproductive indices:
Indices calculated were the copulation index, delivery index and fertility index.
Offspring viability indices:
Viability index between PND 0 to PND 4 was calculated.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The male animal of the control group which was euthanized in moribund condition on treatment day 18 showed moderately reduced spontaneous activity, abnormal breathing and severely increased salivation on the day of sacrifice.
Moving the bedding was observed transiently in 1/10 males and 5/10 females in the high-dose group and increased salivation was noted transiently in 1/10 males in the mid dose, 5/10 males in the high dose and 1/10 females in the low dose and 6/10 females in the high dose. The clinical signs of moving the bedding and increased salivation were observed in a close timely relation to the dose administration and therefore considered to be a sign of discomfort or a local reaction of the test item. They were not considered to beas adverse systemic effects.

The clinical signs hairless area/scratch at different body locations (1/10 males of the low-dose group, 1/10 females of the control, 3/10 females of the low-dose and 3/10 females of the high-dose group), diarrhoea (1/10 males of the high-dose group) and abnormal breathing (2/10 females of the mid-dose group) were seen in single animals without dose dependency or on single observation days and were not considered to be test item-related.
Detailed clinical observation showed no toxicological relevant differences in all male and female groups when compared to the control group.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
During the study a control male was euthanized in moribund condition on treatment day 18. In the histopathological examination, inflammatory cell infiltration was observed in the submucosa of the trachea and at the alveolar duct area of lungs. The microscopic findings suggest that there was unexpected influx of the dosing solution into the respiratory tract. From these findings, the cause of the animal’s morbidity was considered to be a technical error that happened during the oral gavaging procedure.
All remaining animals survived the scheduled study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment with the test item had an effect on the body weight in the male and female high-dose and male mid-dose group during the treatment period.
A statistically significant decrease of body weight was found in high-dose males in the last week of the premating phase and the entire mating/postmating period (up to 14% below control). The body weight at terminal sacrifice showed a statistically significant decrease of 12% below control. The weekly body weight gain was statistically significantly decreased in high-dose males during the treatment period with exception in the second week of mating/postmating. Additionally, a statistically significant decrease in weight gain was noted in the mid-dose male group between premating day 1 to terminal sacrifice (40.57% below control) and the first week of premating and premating day 1 to postmating day 14 (86.84% and 46.31% below control).

In high-dose females, the weekly measurement of body weight showed a decrease between 3.8% and 7.8% compared to control during the lactation phase with statistical significance on lactation day 13 (7.8% below control). The weekly body weight gain was 42.47 % and 39.69% lower than in the control group during lactation, but no statistical significance was found.

No toxicological effect was found in the male and female low-dose and the female mid-dose groups. Body weight development and body weight gain was comparable between test item-treated groups and their respective control group throughout the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The measurement of food consumption showed effects in the male and female high-dose group, which were considered to be related to treatment with the test item.
During the premating phase, the food consumption in high-dose males showed a decrease of 31.11% below control in the first week and 32.55% in the second week of the treatment period. The food consumption in the high-dose female group was statistically significantly decreased during the entire lactation phase when compared to the control group (between 22.97% and 26.63% below control).
The statistically significantly increased mean food consumption between day 0 and day 4 in low-dose females (19.65% above control) was considered to be incidental and of no toxicological relevance.
No test item-related effect on food consumption was found in the male and female low- and mid-dose groups. Mean values were comparable to the respective control group and no specific differences were found.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The test item had no toxicologically relevant effect on haematological and coagulation parameters of selected male and female animals analysed at the end of the treatment period of this study.

Mean values of mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) were statistically significantly decreased in the low-dose and high-dose male group when compared to the control group (low-dose group: 4.62% and 4.99% below control, high-dose group 5.74% and 5.57% below control), but no dose dependency was found between all dose groups. Therefore, no toxicological relevance is considered.

The statistically significant increase of 40.4% above control group for reticulocytes observed in the high-dose male group is considered to be of no toxicological relevance as no dose dependent changes were found between the dose groups and no toxicological effect was seen for red blood cell parameters.

In the absence of dose dependency, the slightly but statistically no significant decreased mean white blood cells value in the male low-dose group of 28.03% and in the high-dose group of 10.80% below the control group is considered to be incidental and without toxicological relevance.

In differential blood cell count, the statistically significant increase of 108.99% in the high-dose male group for monocytes and the statistically significant decrease of 53.84% in the low-dose male group for large unstained cells followed no dose dependency and are therefore considered to be of no toxicological relevance. Additionally, the increased and no statistically significant group mean values for neutrophils in high-dose males (115.95% above control) and 63.12% above control in low-dose males followed no dose dependency and are considered not to be toxicological relevant.

The coagulation parameter prothrombin time, was found with a statistically significant increase in mid-dose males (10.05% above control) and high-dose males (18.08% above control) and is assumed to be of no toxicological relevance as no statistical significant increase for prothrombin time was seen in the female dose groups.

No statistical significant changes were observed for any parameter of haematology and coagulation parameters for all female dose groups. The group mean values were comparable to the respective control group and no differences with toxicological relevance were found.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period, the test item showed effects on clinical biochemical parameters related to the kidney in the male (Urea, Crea) and female (Urea) high-dose group analysed in the selected animals.

Urea was statistically significantly increased in the high-dose male group (41.41% above control), but no dose dependent increase was noted. Additionally, the parameter creatinine (Crea) showed an increase of 47.% above control in high-dose males but without statistical significance. A dose dependent increase was not observed for this parameter. In females, no dose dependency between the low-, mid-, and high-dose group or an increase in the high-dose group were noted for creatinine. Urea showed an increase of 8.47% in the high-dose group, but without statistical significance. The changes observed for the renal clinical chemical parameters correlate to the complex lesions found at histopathological evaluation. Therefore, the slightly increased mean value for Urea in high-dose females cannot fully be excluded as test item-related.

The slight and statistically significant increase of 4.73% above control for albumin in the male low-dose group was considered not to be an effect of the treatment with test item as no such increase was noted in the mid- and high-dose group.

For the liver parameters alanine aminotransferase, aspartate-aminotransferase and alkaline phosphatase, a decrease was noted for all male dose groups when compared to controls with exception of alanine aminotransferase and alkaline phosphatase in the low-dose group. In high-dose males the decrease was up to 21.73% for alanine aminotransferase, 21.41% for aspartate-aminotransferase and 28.24% for alkaline phosphatase (mid-dose group: 36.82% for alkaline phosphatase). A decrease in the mentioned parameters is not considered to be related to the treatment with test item and furthermore, no dose dependent changes with respect to the control group were seen within the male and additionally the female dose groups for (alanine aminotransferase, aspartate-aminotransferase, alkaline phosphatase).

Total bile acids was found with a percentage of 152.76% above control in high-dose and 29.57% above control in mid-dose males. No such increase was found between the female dose groups. Individual total bile acids values in males and females were comparable to the respective control group, with exception of one high level in high-dose male animal, which is considered to have resulted in the high mean value. The differences between the dose groups and the control groups are therefore assumed not to be an effect of treatment with the test item.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The test item had no toxicologically relevant effect on urinary parameters analysed in selected animals at the end of the treatment period of this study.
Findings in single male animals such as ketone in two high-dose animals and slightly higher levels of glucose in two mid-dose animals and one high-dose animal when compared to the control group are considered to be incidental and without relation to the treatment with test item. No specific changes in urinary parameters were noted in all female dose groups when compared to the control group.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
In males and females, no test item-related effect was observed in any of the parameters of the functional observation battery at the end of the treatment period when compared to the respective control group.
Body temperature was observed with a slightly and statistically significant increase in males of the high-dose group in the last week of the treatment period when compared to the control group (38.7 °C in the high-dose group compared to 37.9 °C in the control group). As the increase was slight and no test item-related findings were observed during clinical observation, the statistical difference is considered to be of no toxicological relevance.
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):
Histopathological examination revealed that the test item produced histomorphological changes in kidneys, urinary bladder, ureters, duodenum, jejunum, liver, thyroid glands, bone marrow, thymus and/or adrenal glands of animals that received 750 mg/kg bw/day (high-dose group). This included changes due to primary and secondary effects of the treatment. Qualitatively comparable changes were also observed in kidneys, ureters, urinary bladder of both sexes and in thymus and adrenal glands of males from the mid-dose group (250 mg/kg bw/day).

There were no toxicologically relevant changes in the male and female reproductive organs of animals treated with the test item at a dose of 750 mg/kg bw/day (high-dose group), although increased incidence and group mean severity grade of reduced secretion were found in prostate glands, coagulating glands and seminal vesicles. Reduced secretion was considered to be an alteration secondary to low body weight or due to the stress related to the deteriorlation of general conditions. There were no morphologic abnormalities (such as cellular denegeration or atrophy) in cell- and tissue-construction in these organs, and hence, the reduced secretion without any further indicators of tissue and cellular injury was not considered to be adverse.

In the kidney, urinary bladder and ureter, several degenerative, inflammatory and/or reactive changes were observed in both sexes of the ,mid- and high-dose groups. The treatment related changes observed in the kidney were complex lesions composed of the following changes: increased incidence and severity of tubular basophilia, as well as mixed inflammatory cell infiltrates, interstitial fibrosis, tubular dilatation, tubular casts with mixture of granular, cellular and hyaline, papillary necrosis, pyelitis, pelvic dilatation and hyperplasia of urothelial cells (surface epithelial cells covering the papilla and transitional cells lining the other region of renal calix). Intratubular precipitates and pelvic luminal precipitates were also identified in animals of the high-dose group. In addition, granuloma formed mainly at/around the fornices region was observed in some animals, and foreign body giant cells containing precipitates/ precipitates-like substace were involved in this lesion. The renal lesion was also present in both sexes of the mid-dose group, however, that was of less complexity and severity compared to that of the high-dose group and restricted to the pyelocaliceal region. The degenerative and inflammatory changes, as well as the reactive changes, were also observed in the urinary bladder and ureters of both sexes of the mid- and high-dose group. These included transitional cell hyperplasia accompanied by infiltration of mixed inflammatory cells or mononuclear cells, with occasional hemorrhage, in the the urinary bladder, and transmural inflammation, transitional cell hyperplasia and luminal dilatation in the ureter. In the present study, increased granulopoiesis was observed in both sexes of the high-dose group. This was considered to be a reactive increase associated with inflammatory changes in the urinary system including kidneys, urinary bladder and uteters, and it was not considered to be of an adverse nature.

In the duodenum and jejunum, lipid accumulation in the lamina propria mucosa was observed for both sexes of animals from the high-dose groups. This was recognized as vacuolation or empty space of various sizes, most of which, especially larger vacuolation, were considered to be dilated lymphatic vessels, and some were within the cytoplasm of macrophages or present in the interstitium of the lamina propria.

In the liver, centrilobular hepatocellular hypertrophy was observed for both sexes of the high-dose group. There were no further indicators of liver injury, hence, this change was considered to be of an adaptive nature, most likely to be related to enzyme induction.

In the thyroid glands, diffuse follicular cell hypertrophy was observed for both sexes of the high-dose group. A possible relationship between the liver change was considered, and the changes recorded in the liver and thyroid glands were deemed not to be adverse.

In the adrenal glands and thymus, increase in the incidence and/or severity of vacuolation in zona fasciculata of the adrenal cortex and thymic atrophy was observed in males of the mid-dose group and for both sexes of the high-dose group. Both findings recorded were not considered to be of an adverse nature, but these were considered to be stress-related secondary changes associated with deteriorlation of general conditions, reduction of body weight gain or body weight decrease, or resulting from the lesions in the urinary system.

In conclusion, due to presence of treatment-related changes recorded in kidneys, ureters and urinary bladder for both sexes at 250 mg/kg bw/day and higher, the no-observed-adverse-effect-level (NOAEL) can be established at 62.5 mg/kg bw/day under the conditions of this study. Meanwhile, there were no toxicologically relevant changes in the male and female reproductive organs of animals treated with the test item up to a maximum dose of 750 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Treatment with the test item had no effect on serum T4 levels of parental males. No statistically significant differences for T4 levels and thyroid/parathyroid glands weight were found for any dose group.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There was no effect on the oestrous cycle assessed during the premating period in females of any of the test item-treated groups when compared to the control group. There were no toxicologically relevant considerable differences in the number of normal or abnormal cycles.
Mean cycle length was slightly longer in the high-dose group (5.27 days) when compared to the control (4.25 days). As the increase in the hihg-dose group mean value was slightly above a normal cycle length of 5 days, no test item-related effect is considered.
Reproductive function: sperm measures:
not specified
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
There was no treatment-related effect of the test item on pre-coital interval and duration of gestation observed.

The statistically significantly increased precoital interval (70.8% above control) and duration of gestation (2.6% above control) in the high-dose group was not considered to be a treatment-related effect as no histomorphological changes of toxicological relevance were found in the reproductive organs including testes, epididymides, prostate glands, seminal vesicles, coagulating glands, ovaries, uterus, cervix and vagina from the high-dose group males or females. Additionally, the mean pre-coital interval and duration of gestation were within the range of historical control data.

Mean post implantation loss was observed to be higher in the mid-dose group (post implantation loss: 10.34%) and high-dose group (post implantation loss: 11.97%) compared to the control (post implantation loss: 6.06 %). In the high-dose group, the pre implantation loss was found to be slightly higher (16.20%) than in the control group (13.16%).

Mean percentage of pre and post implantation loss was found to be within the historical control data range for all dose groups and the control group and additionally, no statistical significance was found. An effect of the treatment with test item was therefore not concluded. The pre-natal and post-natal parameters including the number of corpora lutea, implantation sites and alive pups on PND 0, 4 and 13 showed no statistically significant differences between test item-treated groups and the controls.

The copulation index and delivery index was 100% in the control group and in all dose groups.

Fertility index was 100% in the control group, 100% in the low-dose group, 90% in the mid-dose group and 80% in the high-dose group. The slightly lower fertility index in the mid-dose group was caused by 1/10 animals in the mid-dose group and 2/10 animals in the high-dose group, which were not pregnant. At histopathological evaluation no special findings were observed in ovaries, uterus with cervix and vagina. Additionally, no special findings were noted in testes, epididymides, prostate, seminal vesicles and coagulating glands for the high-dose males, which were paired with these females.

Overall, as no histomorphological changes to be of toxicological concern were found in the reproductive organs including ovaries, uterus, cervix and vagina in the two high-dose group and the one mid-dose animals, the lower fertility and viability indexes in mid-dose and/or high-dose females were assumed not to be an effect related to treatment with test item.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
62.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse reproductive effects observed.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
bladder
kidney
ureter
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The survival of pups from PND 0 to PND 4 and from PND 4 to PND 13 was not statistically significantly affected in the test item groups when compared to the control group.
Observed total mortality in the high-dose group of 13.54% between PND 0-4 was achieved by the death of 1/12 pups of dam no. 72 and 7/7 pups of dam no. 80, but no mortality was seen for pups in the high-dose group between PND 4-13. As the mortality was observed for pups of two single dams from the high-dose group and only during the first observation days without statistical significance, no effect of the treatment with test item is considered.
No mortality was observed in the control, low-dose and mid-dose group from PND 0 to PND 4 and from PND 4 to PND 13.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean pup weight on PND 13 was statistically significantly lower in the high-dose group (17.24% below control) when compared to the control group, but no statistically significant difference was found on PND 0 and PND 4. The mean values for mean pup weight on PND 0, 4 and 13 were within the historical control data range for the high-dose group and all other dose groups as well as the control group. Therefore, an effect of the treatment with test item was not assumed.

Total litter mean weight on PND 13 was statistically significantly lower in the high-dose group (26.30% below control) whereas no statistically significant difference to the control was noted for mean male and female litter weight on PND 13. Male litter weight was 38.53% but not statistically significantly below control on PND 13. The litter weight data on PND 0, 4 and 13 were within the historical control data range. An effect of treatment with test item was therefore not assumed.
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:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
Male pups of the high-dose group had a statistically significantly higher absolute (2.63 mm in the high-dose group compared to 2.40 mm in the control group) and relative (1.43 mm in the high-dose group compared to 1.29 mm in the control group) anogenital distance compared to males of the control group. A slight and statistically significantly higher absolute and relative anogenital distance was also seen for female pups (absolute: 1.06 mm in the high-dose group compared to 0.95 mm in the control group; relative: 0.59 mm in the high-dose group compared to 0.52 mm in the control group).
All mean values were within the range of historical control data. Therefore, a relation to treatment with test item on the increase of absolute and relative AGD in high-dose males and females was not assumed.

In the absence of dose dependency the statistically significant increase of cube root pup weight for the male pups (low-dose 1.90, control 1.85) and female pups (low-dose and mid-dose 1.85, control 1.81) is considered to be toxicologically not relevant.
Nipple retention in male pups:
effects observed, non-treatment-related
Description (incidence and severity):
Nipple retention of male pups on PND 12 was seen with a tendency towards a higher mean value between the dose groups, but no statistical significance was found for all dose groups when compared to control (mean high dose up to 0.5, mean control 0.26).
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No gross external abnormalities of toxicological relevance were observed for pups of any of the groups on PND 0-12 or the day of sacrifice/death.

External findings seen in the mid-dose group were a black tail tip (2/10 pups between PND 11-12 and 4/10 pups on day of death from dam number 70). In the high-dose group, for 8/10 pups from one dam hairless area on the back was seen between PND 8-12 and the day of death and additionally, haematoma on the mouth was observed for 1/10 pups on PND 0. No indication of suckling was observed for 2/10 pups from another high-dose female on PND 0 and haematoma on the mouth, neck or back for 4/7 pups of a different high-dose dam on PND 0. The external findings were observed for pups of single dams in the dose groups and therefore considered to be of no toxicological relevance.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Treatment with the test item had no effect on serum T4 levels of pups sacrificed on day 13. Additionally, there were no considerable changes between thyroid/parathyroid glands weight of 13 day old male and female pups in all dose groups and the corresponding control group.

Mean T4 level of female pups on day 13 showed a decrease of 13.8% below control in the mid-dose group and 25.6% in the high-dose group, without achieving statistical significance.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Key result
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
Under the conditions of this study and based on the test item-related finding, the NOAEL is determined to be 62.5 mg/kg bw/day for general toxicity and 750 mg/kg bw/day for reproduction/developmental toxicity.
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Jan - 30 Mar 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developme ntal Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 14-15 weeks old
- Weight at study initiation: males: 337 - 395 g; females: 189 - 233 g
- Housing: housed in groups of 5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages during the premating period for both males and females and during post-mating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female).
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The vehicle corn oil was dried and de-acidified. The test item was weighed into a tared glass vial on a suitable precision balance and the vehicle was added (w/v) to give the appropriate final concentration of the test item. The formulation was alternately vortexed and/or stirred until visual homogeneity was achieved. After homogenization the formulation was overlaid with argon to prevent instability caused by repeated contact of the test item formulation with air.
Based on the results of stability testing, the test item formulations were prepared at least every 11 days. The prepared formulation was stored protected from light and at room temperature.
Formulates were kept under magnetic stirring during the daily administration.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected based on the test item’s characteristics and testing guideline.
- Concentration in vehicle: 15.63, 62.5 and 187.5 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg body weight
- Lot/batch no.: MKCH1635, MKCH6411, 2IC0148
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle as part of a separate GLP study.
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
As the test item was shown to be homogenous (after at least 30 min without stirring), samples were not collected during the study for the investigation of homogeneity and samples were only taken for substance concentration verification in study week 1 (pre-mating period), week 3 (first week of mating), week 5 (gestation) and in the last week of the study (gestation / lactation) from all groups (16 samples).
Concentration analysis: The recoveries observed for the low-dose group was between 93.7% and 99.7% of the nominal value, between 95.6% and 98.2% for the mid-dose dose group and between 94.3% and 99.8% of the nominal value for high-dose group. The mean recoveries observed in the low-, medium- and high-dose groups were 96.9%, 96.6%, and 97.3% of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%.

Details on mating procedure:
- M/F ratio per cage: Mating was performed using a ratio of 1:1 (male to female).
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage.
Duration of treatment / exposure:
Maximum period of 63 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 (PND) 12 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, 7 days/week
Duration of test:
28 days in males and maximum 63 days in females
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
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. In the dose-range finding study, 3 males and 3 females per group were treated orally with the test item at doses of 100, 300 and 1000 mg/kg bw/day for 14 days. Mortality was observed in 1/3 male animals treated with 1000 mg/kg bw/day. Additionally, main test item-related findings at 1000 mg/kg bw/day were macroscopic findings at the kidney/ureter (enlarged/dilated), a tendency for increased mean kidney weight and increased clinical biochemical kidney parameters (Crea, Urea, TP, Alb). No adverse findings were seen in the dose groups treated with 100 and 300 mg/kg bw/day.
Based on this, the highest dose in this study was set as 750 mg/kg bw/day. The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

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 outside the home cage in a standard arena. Inadvertently, the detailed clinical observation was not performed in week 4 for the five randomly selected female animals.
- Clinical observations included: 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.

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 GD 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4, PND 9 and PND 13 along with pups. All animals were weighed directly before termination. Any animals prematurely sacrificed were weighed prior to their sacrifice.

FOOD CONSUMPTION: Yes
- Food consumption: Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the last week of treatment in males and during the last week of lactation in females
- Dose groups that were examined: all dose groups (5 randomly selected animals)


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the time of sacrifice
- Anaesthetic used for blood collection: Yes; ketamine/xylazin
- Animals fasted: Not specified
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the time of sacrifice
- Animals fasted: Not specified
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each group
- Parameters checked in table 2 were examined.

PLASMA/SERUM HORMONES: Yes
- Time of blood sample collection: at the time of sacrifice
- Animals fasted: Not specified
- How many animals: adult males

URINALYSIS: Yes
- Time schedule for collection of urine: at the time of sacrifice
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the last week of the lactation period in 5 randomly selected females. Inadvertently, functional observations of the 5 randomly selected animals were not performed before treatment for female animals, as well as for male animals of the low-, mid- and high-dose groups.
- Dose groups that were examined: all dose groups
- Sensory reactivity to different modalities was assessed: 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

POST-MORTEM EXAMINATIONS: Yes; tables 4 and 5
SACRIFICE
- Male animals: All surviving males were sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days) by using anaesthesia (ketamine/xylazin).
- Maternal animals: Females were sacrificed on the respective PND 13 by using anaesthesia (ketamine/xylazin).

GROSS PATHOLOGY: Yes
Gross necropsy consisted of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4% neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70% ethanol.

ORGAN WEIGHTS: Yes
- The wet weight of the organs (see Table 4) of 5 sacrificed males and 5 females (only lactating females were evaluated) randomly selected from each main group, as well as of all males and females of the recovery groups was recorded as soon as possible. In addition reproductive organs (testes, epididymides, prostate with seminal vesicles and coagulating glands, uterus with cervix and ovaries) of all animals were weighed.
Thyroid/parathyroid glands from 1 pup/sex/litter/group (if possible) (sacrificed on PND 13) and from all adult males and females were preserved. Weight of thyroid/ parathyroid glands was measured after fixation.

HISTOPATHOLOGY: Yes
The tissues indicated in Table 5 from five randomly selected males and females were preserved in 4% neutral-buffered formaldehyde except for eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol.
A full histopathology was carried out on the preserved organs and tissues (Table 5) of the selected animals of the control and high dose group which were sacrificed at the end of the treatment period. A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study or which were euthanised due to morbidity.
For organs and tissues showing treatment-related changes in the high dose group, these examinations were extended to animals of all other dosage groups. Only organs and tissues of the other dosage groups showing changes in the high dose group were examined.
Any gross lesion macroscopically identified was examined microscopically in all animals. Possible discoloration due to the test item was evaluated in the organs of all dose groups.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Blood sampling:
Blood was collected at necropsy and haematological, coagulation and clinical biochemistry parameters examined (for details please refer to "Maternal examinations").
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Anogenital distance of all live rodent pups: Yes

Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Statistics:
A statistical analysis of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry were statistically analysed 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.
Indices:
Indices calculated were the copulation index, delivery index and fertility index. Viability index between PND 0 to PND 4 was calculated.
Historical control data:
Yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The male animal of the control group which was euthanized in moribund condition on treatment day 18 showed moderately reduced spontaneous activity, abnormal breathing and severely increased salivation on the day of sacrifice.

Moving the bedding was observed transiently in 1/10 males and 5/10 females in the high-dose group and increased salivation was noted transiently in 1/10 males in the mid dose, 5/10 males in the high dose and 1/10 females in the low dose and 6/10 females in the high dose. The clinical signs of moving the bedding and increased salivation were observed in a close timely relation to the dose administration and therefore considered to be a sign of discomfort or a local reaction of the test item. They were not considered to be adverse systemic effects.

The clinical signs hairless area/scratch at different body locations (1/10 males of the low-dose group, 1/10 females of the control, 3/10 females of the low-dose and 3/10 females of the high-dose group), diarrhoea (1/10 males of the high-dose group) and abnormal breathing (2/10 females of the mid-dose group) were seen in single animals without dose dependency or on single observation days and were not considered to be test item-related.

Detailed clinical observation showed no toxicological relevant differences in all male and female groups when compared to the control group.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
All dams survived the scheduled study period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
During the study a control male was euthanized in moribund condition on treatment day 18.In the histopathological examination, inflammatory cell infiltration was observed in the submucosa of the trachea and at the alveolar duct area of lungs. The microscopic findings suggest that there was unexpected influx of the dosing solution into the respiratory tract. From these findings, the cause of the animal’s morbidity was considered to be a technical error that happened during the oral gavaging procedure.
All remaining animals survived the scheduled study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment with the test item had an effect on the body weight in the male and female high-dose and male mid-dose group during the treatment period.
A statistically significant decrease of body weight was found in high-dose males in the last week of the premating phase and the entire mating/postmating period (up to 14% below control). The body weight at terminal sacrifice showed a statistically significant decrease of 12% below control. The weekly body weight gain was statistically significantly decreased in high-dose males during the treatment period with exception in the second week of mating/postmating. Additionally, a statistically significant decrease in weight gain was noted in the mid-dose male group between premating day 1 to terminal sacrifice (40.57% below control) and the first week of premating and premating day 1 to postmating day 14 (86.84% and 46.31% below control).

In high-dose females, the weekly measurement of body weight showed a decrease between 3.8% and 7.8% compared to control during the lactation phase with statistical significance on lactation day 13 (7.8% below control). The weekly body weight gain was 42.47 % and 39.69% lower than in the control group during lactation, but no statistical significance was found.

No toxicological effect was found in the male and female low-dose and the female mid-dose groups. Body weight development and body weight gain was comparable between test item-treated groups and their respective control group throughout the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The measurement of food consumption showed effects in the male and female high-dose group, which were considered to be related to treatment with the test item.

During the premating phase, the food consumption in high-dose males showed a decrease of 31.11% below control in the first week and 32.55% in the second week of the treatment period. The food consumption in the high-dose female group was statistically significantly decreased during the entire lactation phase when compared to the control group (between 22.97% and 26.63% below control).

The statistically significantly increased mean food consumption between day 0 and day 4 in low-dose females (19.65% above control) was considered to be incidental and of no toxicological relevance.

No test item-related effect on food consumption was found in the male and female low- and mid-dose groups. Mean values were comparable to the respective control group and no specific differences were found.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The test item had no toxicologically relevant effect on haematological and coagulation parameters of selected male and female animals analysed at the end of the treatment period of this study.

Mean values of mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) were statistically significantly decreased in the low-dose and high-dose male group when compared to the control group (low-dose group: 4.62% and 4.99% below control, high-dose group 5.74% and 5.57% below control), but no dose dependency was found between all dose groups. Therefore, no toxicological relevance is considered.

The statistically significant increase of 40.4% above control group for reticulocytes observed in the high-dose male group is considered to be of no toxicological relevance as no dose dependent changes were found between the dose groups and no toxicological effect was seen for red blood cell parameters.

In the absence of dose dependency, the slightly but statistically no significant decreased mean white blood cells value in the male low-dose group of 28.03% and in the high-dose group of 10.80% below the control group is considered to be incidental and without toxicological relevance.

In differential blood cell count, the statistically significant increase of 108.99% in the high-dose male group for monocytes and the statistically significant decrease of 53.84% in the low-dose male group for large unstained cells followed no dose dependency and are therefore considered to be of no toxicological relevance. Additionally, the increased and no statistically significant group mean values for neutrophils in high-dose males (115.95% above control) and 63.12% above control in low-dose males followed no dose dependency and are considered not to be toxicological relevant.

The coagulation parameter prothrombin time, was found with a statistically significant increase in mid-dose males (10.05% above control) and high-dose males (18.08% above control) and is assumed to be of no toxicological relevance as no statistical significant increase for prothrombin time was seen in the female dose groups.

No statistical significant changes were observed for any parameter of haematology and coagulation parameters for all female dose groups. The group mean values were comparable to the respective control group and no differences with toxicological relevance were found.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
IAt the end of the treatment period, the test item showed effects on clinical biochemical parameters related to the kidney in the male (Urea, Crea) and female (Urea) high-dose group analysed in the selected animals.

Urea was statistically significantly increased in the high-dose male group (41.41% above control), but no dose dependent increase was noted. Additionally, the parameter creatinine (Crea) showed an increase of 47.% above control in high-dose males but without statistical significance. A dose dependent increase was not observed for this parameter. In females, no dose dependency between the low-, mid-, and high-dose group or an increase in the high-dose group were noted for creatinine. Urea showed an increase of 8.47% in the high-dose group, but without statistical significance. The changes observed for the renal clinical chemical parameters correlate to the complex lesions found at histopathological evaluation. Therefore, the slightly increased mean value for Urea in high-dose females cannot fully be excluded as test item-related.

The slight and statistically significant increase of 4.73% above control for albumin in the male low-dose group was considered not to be an effect of the treatment with test item as no such increase was noted in the mid- and high-dose group.

For the liver parameters alanine aminotransferase, aspartate-aminotransferase and alkaline phosphatase, a decrease was noted for all male dose groups when compared to controls with exception of alanine aminotransferase and alkaline phosphatase in the low-dose group. In high-dose males the decrease was up to 21.73% for alanine aminotransferase, 21.41% for aspartate-aminotransferase and 28.24% for alkaline phosphatase (mid-dose group: 36.82% for alkaline phosphatase). A decrease in the mentioned parameters is not considered to be related to the treatment with test item and furthermore, no dose dependent changes with respect to the control group were seen within the male and additionally the female dose groups for (alanine aminotransferase, aspartate-aminotransferase, alkaline phosphatase).

Total bile acids was found with a percentage of 152.76% above control in high-dose and 29.57% above control in mid-dose males. No such increase was found between the female dose groups. Individual total bile acids values in males and females were comparable to the respective control group, with exception of one high level in high-dose male animal, which is considered to have resulted in the high mean value. The differences between the dose groups and the control groups are therefore assumed not to be an effect of treatment with the test item.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The test item had no toxicologically relevant effect on urinary parameters analysed in selected animals at the end of the treatment period of this study.
Findings in single male animals such as ketone in two high-dose animals and slightly higher levels of glucose in two mid-dose animals and one high-dose animal when compared to the control group are considered to be incidental and without relation to the treatment with test item. No specific changes in urinary parameters were noted in all female dose groups when compared to the control group.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
In males and females, no test item-related effect was observed in any of the parameters of the functional observation battery at the end of the treatment period when compared to the respective control group.
Body temperature was observed with a slightly and statistically significant increase in males of the high-dose group in the last week of the treatment period when compared to the control group (38.7 °C in the high-dose group compared to 37.9 °C in the control group). As the increase was slight and no test item-related findings were observed during clinical observation, the statistical difference is considered to be of no toxicological relevance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly higher absolute and relative kidney weight was recorded in males of the high-dose group (absolute 62.52%, relative to body weight 92.59% above control). An increased absolute and relative kidney weight in the female high-dose group did not reach statistical significance (absolute 9.9%, relative to body weight 22.63% above control). The weight changes in the kidneys were considered to be associated with the test item-related complex renal lesions such as tubular basophilia, mixed inflammatory cell infiltrates, interstitial fibrosis, tubular dilatation, tubular casts with mixture of granular, cellular and hyaline, pyelitis, pelvic dilatation and hyperplasia of urothelial cells.

Statistically significant changes were found for the prostate including seminal vesicle and coagulating glands in the male dose groups. Absolute and relative weight to body weight of the accessory reproductive organs were statistically significantly decreased in the low- (absolute 15.59%, relative 17.56% below control) and high-dose group (absolute 30.63%, relative 21.48% below control), whereas in the mid-dose group the decrease in mean absolute and relative weight was found without statistical significance. The absolute and relative thymus weight in males showed a tendency towards a decrease between the dose groups and was statistically significantly decreased in the high-dose group (absolute 40.47% below control). In females, no statistical significant changes were found for all dose groups, but the absolute and relative mean weight showed a tendency to decrease when compared to the control (absolute 29.58% and relative 20.80% below control) in the female high-dose group. Following consideration of the histopathological evaluation, the weight changes of the thymus and the accessory male reproductive organs were deemed to be an alteration secondary to low body weight (statistically significantly decreased in high-dose males and high-dose females (12.28% and 7.8% below control at necropsy) or due to the stress related to the deterioration of general conditions.

Statistically significant changes from the control were found for pituitary glands in the mid-dose male group (relative weight to body weight: 19.50% above control), heart in the high-dose male group (relative weight to body weight: 14.84% above control) and in high-dose females for brain weight (absolute weight 7.77% below control). In the absence of dose dependent changes and test item-related findings observed at histopathological evaluation, it was considered that the changes for pituitary glands, heart and brain were of no toxicological relevance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were gross lesions observed in the male and female high-dose groups and the female mid-dose group, which were considered to be an effect of the test item. Adverse test item-related findings were noted in kidney, urinary bladder and ureter in the high-dose groups and the female mid-dose group (enlarged ureter in 1/10 mid-dose females).
For one control male, which was euthanized for animal welfare reasons in a moribund condition on treatment day 18, was observed with dark red fluid in the trachea, red coloured lung and dark thymus. Histopathologically, mixed inflammatory cell infiltration was observed in the submucosa of the trachea and at the alveolar duct area of lungs. The findings in the respiratory tract indicate a technical error during the oral gavage as the cause of the moribund condition.

Test item-related findings were seen in the high-dose male group in the kidney, ureter and urinary bladder. Findings of the kidney were fluid yellow content in 1/10 high-dose males, dilatation of the right kidney in 1/10 high-dose males, enlarged kidney for 5/10 animals and pelvis dilatation for 1/10 males. The ureter were filled with yellow or white fluid (for 2 high-dose males) and enlarged/dilated in 5/10 high-dose males. For 5/10 high-dose males the urinary bladder was found with a thickened wall. In females, test item-related findings were noted in the kidney of 1/10 high-dose females (right enlarged, green), for the ureter of 2/10 high-dose females (dilatation) and 1/10 mid-dose female animals (enlarged). The thickened wall of the urinary bladder was noted in 3/10 high-dose females and the organ was enlarged for 2/10 high-dose females.

Further findings in the female high-dose group were red coloured salivary glands and dark red/brown coloured axillary lymph nodes for one high-dose female. These findings were considered not to be related to the treatment with test item, as they were seen in one single animal from the high-dose group or were also found in all other female dose groups (1/10 low-dose animals, 2/10 mid-dose animals) and the control group (2/10 animals). The findings of brown coloured liver (one low-dose female), slightly enlarged adrenal glands (one low-dose female) and small right uterus (one control female) were considered to be incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examination revealed that the test item produced histomorphological changes in kidneys, urinary bladder, ureters, duodenum, jejunum, liver, thyroid glands, bone marrow, thymus and/or adrenal glands of animals that received 750 mg/kg bw/day (high-dose group). This included changes due to primary and secondary effects of the treatment. Qualitatively comparable changes were also observed in kidneys, ureters, urinary bladder of both sexes and in thymus and adrenal glands of males from the mid-dose group (250 mg/kg bw/day).

There were no toxicologically relevant changes in the male and female reproductive organs of animals treated with the test item at a dose of 750 mg/kg bw/day (high-dose group), although increased incidence and group mean severity grade of reduced secretion were found in prostate glands, coagulating glands and seminal vesicles. Reduced secretion was considered to be an alteration secondary to low body weight or due to the stress related to the deteriorlation of general conditions. There were no morphologic abnormalities (such as cellular denegeration or atrophy) in cell- and tissue-construction in these organs, and hence, the reduced secretion without any further indicators of tissue and cellular injury was not considered to be adverse.

In the kidney, urinary bladder and ureter, several degenerative, inflammatory and/or reactive changes were observed in both sexes of the ,mid- and high-dose groups. The treatment related changes observed in the kidney were complex lesions composed of the following changes: increased incidence and severity of tubular basophilia, as well as mixed inflammatory cell infiltrates, interstitial fibrosis, tubular dilatation, tubular casts with mixture of granular, cellular and hyaline, papillary necrosis, pyelitis, pelvic dilatation and hyperplasia of urothelial cells (surface epithelial cells covering the papilla and transitional cells lining the other region of renal calix). Intratubular precipitates and pelvic luminal precipitates were also identified in animals of the high-dose group. In addition, granuloma formed mainly at/around the fornices region was observed in some animals, and foreign body giant cells containing precipitates/ precipitates-like substace were involved in this lesion. The renal lesion was also present in both sexes of the mid-dose group, however, that was of less complexity and severity compared to that of the high-dose group and restricted to the pyelocaliceal region. The degenerative and inflammatory changes, as well as the reactive changes, were also observed in the urinary bladder and ureters of both sexes of the mid- and high-dose group. These included transitional cell hyperplasia accompanied by infiltration of mixed inflammatory cells or mononuclear cells, with occasional hemorrhage, in the the urinary bladder, and transmural inflammation, transitional cell hyperplasia and luminal dilatation in the ureter. In the present study, increased granulopoiesis was observed in both sexes of the high-dose group. This was considered to be a reactive increase associated with inflammatory changes in the urinary system including kidneys, urinary bladder and uteters, and it was not considered to be of an adverse nature.

In the duodenum and jejunum, lipid accumulation in the lamina propria mucosa was observed for both sexes of animals from the high-dose groups. This was recognized as vacuolation or empty space of various sizes, most of which, especially larger vacuolation, were considered to be dilated lymphatic vessels, and some were within the cytoplasm of macrophages or present in the interstitium of the lamina propria.

In the liver, centrilobular hepatocellular hypertrophy was observed for both sexes of the high-dose group. There were no further indicators of liver injury, hence, this change was considered to be of an adaptive nature, most likely to be related to enzyme induction.

In the thyroid glands, diffuse follicular cell hypertrophy was observed for both sexes of the high-dose group. A possible relationship between the liver change was considered, and the changes recorded in the liver and thyroid glands were deemed not to be adverse.

In the adrenal glands and thymus, increase in the incidence and/or severity of vacuolation in zona fasciculata of the adrenal cortex and thymic atrophy was observed in males of the mid-dose group and for both sexes of the high-dose group. Both findings recorded were not considered to be of an adverse nature, but these were considered to be stress-related secondary changes associated with deteriorlation of general conditions, reduction of body weight gain or body weight decrease, or resulting from the lesions in the urinary system.

In conclusion, due to presence of treatment-related changes recorded in kidneys, ureters and urinary bladder for both sexes at 250 mg/kg bw/day and higher, the no-observed-adverse-effect-level (NOAEL) can be established at 62.5 mg/kg bw/day under the conditions of this study. Meanwhile, there were no toxicologically relevant changes in the male and female reproductive organs of animals treated with the test item up to a maximum dose of 750 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Treatment with the test item had no effect on serum T4 levels of parental males.
Number of abortions:
no effects observed
Description (incidence and severity):
The delivery index was 100% in the control and in all dose groups.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Mean post implantation loss was observed to be higher in the mid-dose group (post implantation loss: 10.34%) and high-dose group (post implantation loss: 11.97%) compared to the control (post implantation loss: 6.06%). In the high-dose group, the pre implantation loss was found to be slightly higher (16.20%) than in the control group (13.16%).

Mean percentage of pre and post implantation loss was found to be within the historical control data range for all dose groups and the control group and additionally, no statistical significance was found. An effect of the treatment with test item was therefore not concluded.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Observed total mortality in the high-dose group of 13.54% between PND 0-4 was achieved by the death of 1/12 pups of dam no. 72 and 7/7 pups of dam no. 80, but no mortality was seen for pups in the high-dose group between PND 4-13. As the mortality was observed for pups of two single dams from the high-dose group and only during the first observation days without statistical significance, no effect of the treatment with test item is considered.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There was no treatment-related effect of the test item on pre-coital interval and duration of gestation observed.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
The delivery index was 100% in the control group and in all dose groups.
Fertility index was 100% in the control group, 100% in the low-dose group, 90% in the mid-dose group and 80% in the high-dose group. The slightly lower fertility index in the mid-dose group was caused by 1/10 animals in the mid-dose group and 2/10 animals in the high-dose group, which were not pregnant. At histopathological evaluation no special findings were observed in ovaries, uterus with cervix and vagina. Additionally, no special findings were noted in testes, epididymides, prostate, seminal vesicles and coagulating glands for the high-dose males, which were paired with these females.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
62.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: urinary (kidney, bladder, ureter)
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean pup weight on PND 13 was statistically significantly lower in the high-dose group (17.24% below control) when compared to the control group, but no statistically significant difference was found on PND 0 and PND 4. The mean values for mean pup weight on PND 0, 4 and 13 were within the historical control data range for the high-dose group and all other dose groups as well as the control group. Therefore, an effect of the treatment with test item was not assumed.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
One out of 11 pups of a control dam and 2/7 pups of another control dam were found dead on PND 0. Two out of 10 pups of a mid-dose dam were found dead on PND 0. In the high-dose group, 1/12 pups from one dam and and 5/7 pups from another dam (number 80) were not found on PND 0 and additionally 2/7 pups of dam no. 80 on PND 3sex. Death of pups was noted in dose groups as well as the control group and considered to be of no toxicological relevance.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The number of male and female pups and sex ratio (m/f) on PND 0, 4 and 13 were not statistically significantly different in test item groups compared to the control group. No treatment-related effect on these parameters is concluded.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
The litter parameters including the total number of pups was not statistically significantly different in test item groups compared to the control group. No treatment-related effect on this parameter is concluded.
Total litter mean weight on PND 13 was statistically significantly lower in the high-dose group (26.30% below control) whereas no statistically significant difference to the control was noted for mean male and female litter weight on PND 13. Male litter weight was 38.53% but not statistically significantly below control on PND 13. The litter weight data on PND 0, 4 and 13 were within the historical control data range. An effect of treatment with test item was therefore not assumed.
Anogenital distance of all rodent fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Male pups of the high-dose group had a statistically significantly higher absolute (2.63 mm in the high-dose group compared to 2.40 mm in the control group) and relative (1.43 mm in the high-dose group compared to 1.29 mm in the control group) anogenital distance compared to males of the control group. A slight and statistically significantly higher absolute and relative anogenital distance was also seen for female pups (absolute: 1.06 mm in the high-dose group compared to 0.95 mm in the control group; relative: 0.59 mm in the high-dose group compared to 0.52 mm in the control group).
All mean values were within the range of historical control data. Therefore, a relation to treatment with test item on the increase of absolute and relative AGD in high-dose males and females was not assumed.
In the absence of dose dependency the statistically significant increase of cube root pup weight for the male pups (low dose 1.90, control 1.85) and female pups (low-dose and mid-dose 1.85, control 1.81) is considered to be toxicologically not relevant.
Changes in postnatal survival:
effects observed, non-treatment-related
Description (incidence and severity):
Two out of 7 pups from a high-dose dam were not found on PND 3. This was considered to be of no toxicological relevance.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No gross external abnormalities of toxicological relevance were observed for pups of any of the groups on PND 0-12 or the day of sacrifice/death.
External findings seen in the mid-dose group were a black tail tip (2/10 pups between PND 11-12 and 4/10 pups on day of death from dam number 70). In the high-dose group, for 8/10 pups from one dam hairless area on the back was seen between PND 8-12 and the day of death and additionally, haematoma on the mouth was observed for 1/10 pups on PND 0. No indication of suckling was observed for 2/10 pups from another high-dose female on PND 0 and haematoma on the mouth, neck or back for 4/7 pups of a different high-dose dam on PND 0. The external findings were observed for pups of single dams in the dose groups and therefore considered to be of no toxicological relevance.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Nipple retention of male pups on PND 12 was seen with a tendency towards a higher mean value between the dose groups, but no statistical significance was found for all dose groups when compared to control (mean high dose up to 0.5, mean control 0.26).

Treatment with the test item had no effect on serum T4 levels of pups sacrificed on day 13. Additionally, there were no considerable changes between thyroid/parathyroid glands weight of 13 day old male and female pups in all dose groups and the corresponding control group.
Mean T4 level of female pups on day 13 showed a decrease of 13.8 % below control in the mid-dose group and 25.6 % in the high-dose group, without achieving statistical significance.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No developmental effects observed.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Under the conditions of this study and based on the test item-related finding, the NOAEL is determined to be 62.5 mg/kg bw/day for maternal toxicity and 750 mg/kg bw/day for developmental toxicity.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Triethoxy(vinyl)silane
EC Number:
201-081-7
EC Name:
Triethoxy(vinyl)silane
Cas Number:
78-08-0
Molecular formula:
C8H18O3Si
IUPAC Name:
triethoxy(vinyl)silane
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 14-15 weeks old
- Weight at study initiation: males: 337 - 395 g; females: 189 - 233 g
- Housing: housed in groups of 5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages during the premating period for both males and females and during post-mating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female).
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8, ad libitum
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY:
Certificates of food and water were available and archived.

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The vehicle corn oil was dried and de-acidified. The test item was weighed into a tared glass vial on a suitable precision balance and the vehicle was added (w/v) to give the appropriate final concentration of the test item. The formulation was alternately vortexed and/or stirred until visual homogeneity was achieved. After homogenization the formulation was overlaid with argon to prevent instability caused by repeated contact of the test item formulation with air.
Based on the results of stability testing, the test item formulations were prepared at least every 11 days. The prepared formulation was stored protected from light and at room temperature.
Formulates were kept under magnetic stirring during the daily administration.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected based on the test item’s characteristics and testing guideline.
- Concentration in vehicle: 15.63, 62.5 and 187.5 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg body weight
- Lot/batch no.: MKCH1635, MKCH6411, 2IC0148
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle as part of a separate GLP study.
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
As the test item was shown to be homogenous (after at least 30 min without stirring), samples were not collected during the study for the investigation of homogeneity and samples were only taken for substance concentration verification in study week 1 (pre-mating period), week 3 (first week of mating), week 5 (gestation) and in the last week of the study (gestation / lactation) from all groups (16 samples).
Concentration analysis: The recoveries observed for the low-dose group was between 93.7% and 99.7% of the nominal value, between 95.6% and 98.2% for the mid-dose dose group and between 94.3% and 99.8% of the nominal value for high-dose group. The mean recoveries observed in the low-, medium- and high-dose groups were 96.9%, 96.6%, and 97.3% of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%.

Duration of treatment / exposure:
Maximum period of 63 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 (PND) 12 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, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
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. In the dose-range finding study, 3 males and 3 females per group were treated orally with the test item at doses of 100, 300 and 1000 mg/kg bw/day for 14 days. Mortality was observed in 1/3 male animals treated with 1000 mg/kg bw/day. Additionally, main test item-related findings at 1000 mg/kg bw/day were macroscopic findings at the kidney/ureter (enlarged/dilated), a tendency for increased mean kidney weight and increased clinical biochemical kidney parameters (Crea, Urea, TP, Alb). No adverse findings were seen in the dose groups treated with 100 and 300 mg/kg bw/day.
Based on this, the highest dose in this study was set as 750 mg/kg bw/day. The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.
Positive control:
None

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. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

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 outside the home cage in a standard arena. Inadvertently, the detailed clinical observation was not performed in week 4 for the five randomly selected female animals.
- Clinical observations included: 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.

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 GD 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4, PND 9 and PND 13 along with pups. All animals were weighed directly before termination. Any animals prematurely sacrificed were weighed prior to their sacrifice.

FOOD CONSUMPTION: Yes
- Food consumption: Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the last week of treatment in males and during the last week of lactation in females
- Dose groups that were examined: all dose groups (5 randomly selected animals)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the time of sacrifice
- Anaesthetic used for blood collection: Yes; ketamine/xylazin
- Animals fasted: Not specified
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the time of sacrifice
- Animals fasted: Not specified
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each group
- Parameters checked in table 2 were examined.

PLASMA/SERUM HORMONES: Yes
- Time of blood sample collection: at the time of sacrifice
- Animals fasted: Not specified
- How many animals: adult males

URINALYSIS: Yes
- Time schedule for collection of urine: at the time of sacrifice
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the last week of the lactation period in 5 randomly selected females. Inadvertently, functional observations of the 5 randomly selected animals were not performed before treatment for female animals, as well as for male animals of the low-, mid- and high-dose groups.
- Dose groups that were examined: all dose groups
- Sensory reactivity to different modalities was assessed: 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:
OESTROUS CYCLE
Oestrous cycles were monitored before treatment started to select for the study females with regular oestrous cyclicity. Vaginal smears were examined daily from the beginning of the treatment period until evidence of mating.
Vaginal smears were also examined on the day of necropsy to determine the stage of oestrous cycle.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Gross necropsy consisted of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4% neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70% ethanol.

ORGAN WEIGHTS: Yes
- The wet weight of the organs (see Table 4) of 5 sacrificed males and 5 females (only lactating females were evaluated) randomly selected from each main group, as well as of all males and females of the recovery groups was recorded as soon as possible. In addition reproductive organs (testes, epididymides, prostate with seminal vesicles and coagulating glands, uterus with cervix and ovaries) of all animals were weighed.
Thyroid/parathyroid glands from 1 pup/sex/litter/group (if possible) (sacrificed on PND 13) and from all adult males and females were preserved. Weight of thyroid/ parathyroid glands was measured after fixation.

HISTOPATHOLOGY: Yes
The tissues indicated in Table 5 from five randomly selected males and females were preserved in 4% neutral-buffered formaldehyde except for eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol.
A full histopathology was carried out on the preserved organs and tissues (Table 5) of the selected animals of the control and high dose group which were sacrificed at the end of the treatment period. A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study or which were euthanised due to morbidity.
For organs and tissues showing treatment-related changes in the high dose group, these examinations were extended to animals of all other dosage groups. Only organs and tissues of the other dosage groups showing changes in the high dose group were examined.
Any gross lesion macroscopically identified was examined microscopically in all animals. Possible discoloration due to the test item was evaluated in the organs of all dose groups.
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides.
Optional endpoint(s):
None
Other examinations:
None
Statistics:
A statistical analysis of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a nonparametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The male animal of the control group which was euthanized in moribund condition on treatment day 18 showed moderately reduced spontaneous activity, abnormal breathing and severely increased salivation on the day of sacrifice.

Moving the bedding was observed transiently in 1/10 males and 5/10 females in the high-dose group and increased salivation was noted transiently in 1/10 males in the mid dose, 5/10 males in the high dose and 1/10 females in the low dose and 6/10 females in the high dose. The clinical signs of moving the bedding and increased salivation were observed in a close timely relation to the dose administration and therefore considered to be a sign of discomfort or a local reaction of the test item. They were not considered to be adverse systemic effects.

The clinical signs hairless area/scratch at different body locations (1/10 males of the low-dose group, 1/10 females of the control, 3/10 females of the low-dose and 3/10 females of the high-dose group), diarrhoea (1/10 males of the high-dose group) and abnormal breathing (2/10 females of the mid-dose group) were seen in single animals without dose dependency or on single observation days and were not considered to be test item-related.

Detailed clinical observation showed no toxicological relevant differences in all male and female groups when compared to the control group.

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
During the study a control male was euthanized in moribund condition on treatment day 18.In the histopathological examination, inflammatory cell infiltration was observed in the submucosa of the trachea and at the alveolar duct area of lungs. The microscopic findings suggest that there was unexpected influx of the dosing solution into the respiratory tract. From these findings, the cause of the animal’s morbidity was considered to be a technical error that happened during the oral gavaging procedure.
All remaining animals survived the scheduled study period.

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment with the test item had an effect on the body weight in the male and female high-dose and male mid-dose group during the treatment period.
A statistically significant decrease of body weight was found in high-dose males in the last week of the premating phase and the entire mating/postmating period (up to 14% below control). The body weight at terminal sacrifice showed a statistically significant decrease of 12% below control. The weekly body weight gain was statistically significantly decreased in high-dose males during the treatment period with exception in the second week of mating/postmating. Additionally, a statistically significant decrease in weight gain was noted in the mid-dose male group between premating day 1 to terminal sacrifice (40.57% below control) and the first week of premating and premating day 1 to postmating day 14 (86.84% and 46.31% below control).

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).

In high-dose females, the weekly measurement of body weight showed a decrease between 3.8% and 7.8% compared to control during the lactation phase with statistical significance on lactation day 13 (7.8% below control). The weekly body weight gain was 42.47 % and 39.69% lower than in the control group during lactation, but no statistical significance was found.

No toxicological effect was found in the male and female low-dose and the female mid-dose groups. Body weight development and body weight gain was comparable between test item-treated groups and their respective control group throughout the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The measurement of food consumption showed effects in the male and female high-dose group, which were considered to be related to treatment with the test item.

During the premating phase, the food consumption in high-dose males showed a decrease of 31.11% below control in the first week and 32.55% in the second week of the treatment period. The food consumption in the high-dose female group was statistically significantly decreased during the entire lactation phase when compared to the control group (between 22.97% and 26.63% below control).

The statistically significantly increased mean food consumption between day 0 and day 4 in low-dose females (19.65% above control) was considered to be incidental and of no toxicological relevance.

No test item-related effect on food consumption was found in the male and female low- and mid-dose groups. Mean values were comparable to the respective control group and no specific differences were found.

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The test item had no toxicologically relevant effect on haematological and coagulation parameters of selected male and female animals analysed at the end of the treatment period of this study.

Mean values of mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) were statistically significantly decreased in the low-dose and high-dose male group when compared to the control group (low-dose group: 4.62% and 4.99% below control, high-dose group 5.74% and 5.57% below control), but no dose dependency was found between all dose groups. Therefore, no toxicological relevance is considered.

The statistically significant increase of 40.4% above control group for reticulocytes observed in the high-dose male group is considered to be of no toxicological relevance as no dose dependent changes were found between the dose groups and no toxicological effect was seen for red blood cell parameters.

In the absence of dose dependency, the slightly but statistically no significant decreased mean white blood cells value in the male low-dose group of 28.03% and in the high-dose group of 10.80% below the control group is considered to be incidental and without toxicological relevance.

In differential blood cell count, the statistically significant increase of 108.99% in the high-dose male group for monocytes and the statistically significant decrease of 53.84% in the low-dose male group for large unstained cells followed no dose dependency and are therefore considered to be of no toxicological relevance. Additionally, the increased and no statistically significant group mean values for neutrophils in high-dose males (115.95% above control) and 63.12% above control in low-dose males followed no dose dependency and are considered not to be toxicological relevant.

The coagulation parameter prothrombin time, was found with a statistically significant increase in mid-dose males (10.05% above control) and high-dose males (18.08% above control) and is assumed to be of no toxicological relevance as no statistical significant increase for prothrombin time was seen in the female dose groups.

No statistical significant changes were observed for any parameter of haematology and coagulation parameters for all female dose groups. The group mean values were comparable to the respective control group and no differences with toxicological relevance were found.

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period, the test item showed effects on clinical biochemical parameters related to the kidney in the male (Urea, Crea) and female (Urea) high-dose group analysed in the selected animals.

Urea was statistically significantly increased in the high-dose male group (41.41% above control), but no dose dependent increase was noted. Additionally, the parameter creatinine (Crea) showed an increase of 47.% above control in high-dose males but without statistical significance. A dose dependent increase was not observed for this parameter. In females, no dose dependency between the low-, mid-, and high-dose group or an increase in the high-dose group were noted for creatinine. Urea showed an increase of 8.47% in the high-dose group, but without statistical significance. The changes observed for the renal clinical chemical parameters correlate to the complex lesions found at histopathological evaluation. Therefore, the slightly increased mean value for Urea in high-dose females cannot fully be excluded as test item-related.

The slight and statistically significant increase of 4.73% above control for albumin in the male low-dose group was considered not to be an effect of the treatment with test item as no such increase was noted in the mid- and high-dose group.

For the liver parameters alanine aminotransferase, aspartate-aminotransferase and alkaline phosphatase, a decrease was noted for all male dose groups when compared to controls with exception of alanine aminotransferase and alkaline phosphatase in the low-dose group. In high-dose males the decrease was up to 21.73% for alanine aminotransferase, 21.41% for aspartate-aminotransferase and 28.24% for alkaline phosphatase (mid-dose group: 36.82% for alkaline phosphatase). A decrease in the mentioned parameters is not considered to be related to the treatment with test item and furthermore, no dose dependent changes with respect to the control group were seen within the male and additionally the female dose groups for (alanine aminotransferase, aspartate-aminotransferase, alkaline phosphatase).

Total bile acids was found with a percentage of 152.76% above control in high-dose and 29.57% above control in mid-dose males. No such increase was found between the female dose groups. Individual total bile acids values in males and females were comparable to the respective control group, with exception of one high level in high-dose male animal, which is considered to have resulted in the high mean value. The differences between the dose groups and the control groups are therefore assumed not to be an effect of treatment with the test item.

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The test item had no toxicologically relevant effect on urinary parameters analysed in selected animals at the end of the treatment period of this study.
Findings in single male animals such as ketone in two high-dose animals and slightly higher levels of glucose in two mid-dose animals and one high-dose animal when compared to the control group are considered to be incidental and without relation to the treatment with test item. No specific changes in urinary parameters were noted in all female dose groups when compared to the control group.

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
In males and females, no test item-related effect was observed in any of the parameters of the functional observation battery at the end of the treatment period when compared to the respective control group.
Body temperature was observed with a slightly and statistically significant increase in males of the high-dose group in the last week of the treatment period when compared to the control group (38.7 °C in the high-dose group compared to 37.9 °C in the control group). As the increase was slight and no test item-related findings were observed during clinical observation, the statistical difference is considered to be of no toxicological relevance.

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly higher absolute and relative kidney weight was recorded in males of the high-dose group (absolute 62.52%, relative to body weight 92.59% above control). An increased absolute and relative kidney weight in the female high-dose group did not reach statistical significance (absolute 9.9%, relative to body weight 22.63% above control). The weight changes in the kidneys were considered to be associated with the test item-related complex renal lesions such as tubular basophilia, mixed inflammatory cell infiltrates, interstitial fibrosis, tubular dilatation, tubular casts with mixture of granular, cellular and hyaline, pyelitis, pelvic dilatation and hyperplasia of urothelial cells.

Statistically significant changes were found for the prostate including seminal vesicle and coagulating glands in the male dose groups. Absolute and relative weight to body weight of the accessory reproductive organs were statistically significantly decreased in the low- (absolute 15.59%, relative 17.56% below control) and high-dose group (absolute 30.63%, relative 21.48% below control), whereas in the mid-dose group the decrease in mean absolute and relative weight was found without statistical significance. The absolute and relative thymus weight in males showed a tendency towards a decrease between the dose groups and was statistically significantly decreased in the high-dose group (absolute 40.47% below control). In females, no statistical significant changes were found for all dose groups, but the absolute and relative mean weight showed a tendency to decrease when compared to the control (absolute 29.58% and relative 20.80% below control) in the female high-dose group. Following consideration of the histopathological evaluation, the weight changes of the thymus and the accessory male reproductive organs were deemed to be an alteration secondary to low body weight (statistically significantly decreased in high-dose males and high-dose females (12.28% and 7.8% below control at necropsy) or due to the stress related to the deterioration of general conditions.

Statistically significant changes from the control were found for pituitary glands in the mid-dose male group (relative weight to body weight: 19.50% above control), heart in the high-dose male group (relative weight to body weight: 14.84% above control) and in high-dose females for brain weight (absolute weight 7.77% below control). In the absence of dose dependent changes and test item-related findings observed at histopathological evaluation, it was considered that the changes for pituitary glands, heart and brain were of no toxicological relevance.

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were gross lesions observed in the male and female high-dose groups and the female mid-dose group, which were considered to be an effect of the test item. Adverse test item-related findings were noted in kidney, urinary bladder and ureter in the high-dose groups and the female mid-dose group (enlarged ureter in 1/10 mid-dose females).
For one control male, which was euthanized for animal welfare reasons in a moribund condition on treatment day 18, was observed with dark red fluid in the trachea, red coloured lung and dark thymus. Histopathologically, mixed inflammatory cell infiltration was observed in the submucosa of the trachea and at the alveolar duct area of lungs. The findings in the respiratory tract indicate a technical error during the oral gavage as the cause of the moribund condition.

Test item-related findings were seen in the high-dose male group in the kidney, ureter and urinary bladder. Findings of the kidney were fluid yellow content in 1/10 high-dose males, dilatation of the right kidney in 1/10 high-dose males, enlarged kidney for 5/10 animals and pelvis dilatation for 1/10 males. The ureter were filled with yellow or white fluid (for 2 high-dose males) and enlarged/dilated in 5/10 high-dose males. For 5/10 high-dose males the urinary bladder was found with a thickened wall. In females, test item-related findings were noted in the kidney of 1/10 high-dose females (right enlarged, green), for the ureter of 2/10 high-dose females (dilatation) and 1/10 mid-dose female animals (enlarged). The thickened wall of the urinary bladder was noted in 3/10 high-dose females and the organ was enlarged for 2/10 high-dose females.

Further findings in the female high-dose group were red coloured salivary glands and dark red/brown coloured axillary lymph nodes for one high-dose female. These findings were considered not to be related to the treatment with test item, as they were seen in one single animal from the high-dose group or were also found in all other female dose groups (1/10 low-dose animals, 2/10 mid-dose animals) and the control group (2/10 animals). The findings of brown coloured liver (one low-dose female), slightly enlarged adrenal glands (one low-dose female) and small right uterus (one control female) were considered to be incidental.

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examination revealed that the test item produced histomorphological changes in kidneys, urinary bladder, ureters, duodenum, jejunum, liver, thyroid glands, bone marrow, thymus and/or adrenal glands of animals that received 750 mg/kg bw/day (high-dose group). This included changes due to primary and secondary effects of the treatment. Qualitatively comparable changes were also observed in kidneys, ureters, urinary bladder of both sexes and in thymus and adrenal glands of males from the mid-dose group (250 mg/kg bw/day).

There were no toxicologically relevant changes in the male and female reproductive organs of animals treated with the test item at a dose of 750 mg/kg bw/day (high-dose group), although increased incidence and group mean severity grade of reduced secretion were found in prostate glands, coagulating glands and seminal vesicles. Reduced secretion was considered to be an alteration secondary to low body weight or due to the stress related to the deteriorlation of general conditions. There were no morphologic abnormalities (such as cellular denegeration or atrophy) in cell- and tissue-construction in these organs, and hence, the reduced secretion without any further indicators of tissue and cellular injury was not considered to be adverse.

In the kidney, urinary bladder and ureter, several degenerative, inflammatory and/or reactive changes were observed in both sexes of the ,mid- and high-dose groups. The treatment related changes observed in the kidney were complex lesions composed of the following changes: increased incidence and severity of tubular basophilia, as well as mixed inflammatory cell infiltrates, interstitial fibrosis, tubular dilatation, tubular casts with mixture of granular, cellular and hyaline, papillary necrosis, pyelitis, pelvic dilatation and hyperplasia of urothelial cells (surface epithelial cells covering the papilla and transitional cells lining the other region of renal calix). Intratubular precipitates and pelvic luminal precipitates were also identified in animals of the high-dose group. In addition, granuloma formed mainly at/around the fornices region was observed in some animals, and foreign body giant cells containing precipitates/ precipitates-like substace were involved in this lesion. The renal lesion was also present in both sexes of the mid-dose group, however, that was of less complexity and severity compared to that of the high-dose group and restricted to the pyelocaliceal region. The degenerative and inflammatory changes, as well as the reactive changes, were also observed in the urinary bladder and ureters of both sexes of the mid- and high-dose group. These included transitional cell hyperplasia accompanied by infiltration of mixed inflammatory cells or mononuclear cells, with occasional hemorrhage, in the the urinary bladder, and transmural inflammation, transitional cell hyperplasia and luminal dilatation in the ureter. In the present study, increased granulopoiesis was observed in both sexes of the high-dose group. This was considered to be a reactive increase associated with inflammatory changes in the urinary system including kidneys, urinary bladder and uteters, and it was not considered to be of an adverse nature.

In the duodenum and jejunum, lipid accumulation in the lamina propria mucosa was observed for both sexes of animals from the high-dose groups. This was recognized as vacuolation or empty space of various sizes, most of which, especially larger vacuolation, were considered to be dilated lymphatic vessels, and some were within the cytoplasm of macrophages or present in the interstitium of the lamina propria.

In the liver, centrilobular hepatocellular hypertrophy was observed for both sexes of the high-dose group. There were no further indicators of liver injury, hence, this change was considered to be of an adaptive nature, most likely to be related to enzyme induction.

In the thyroid glands, diffuse follicular cell hypertrophy was observed for both sexes of the high-dose group. A possible relationship between the liver change was considered, and the changes recorded in the liver and thyroid glands were deemed not to be adverse.

In the adrenal glands and thymus, increase in the incidence and/or severity of vacuolation in zona fasciculata of the adrenal cortex and thymic atrophy was observed in males of the mid-dose group and for both sexes of the high-dose group. Both findings recorded were not considered to be of an adverse nature, but these were considered to be stress-related secondary changes associated with deteriorlation of general conditions, reduction of body weight gain or body weight decrease, or resulting from the lesions in the urinary system.

In conclusion, due to presence of treatment-related changes recorded in kidneys, ureters and urinary bladder for both sexes at 250 mg/kg bw/day and higher, the no-observed-adverse-effect-level (NOAEL) can be established at 62.5 mg/kg bw/day under the conditions of this study. Meanwhile, there were no toxicologically relevant changes in the male and female reproductive organs of animals treated with the test item up to a maximum dose of 750 mg/kg bw/day.

No detailed description of the histopathology findings are available in the draft report. Further details will be provided once the final report is available.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment with the test item had no effect on serum T4 levels of parental males.

There was no effect on the oestrous cycle assessed during the premating period in females of any of the test item-treated groups when compared to the control group. There were no toxicologically relevant considerable differences in the number of normal or abnormal cycles.
Mean cycle length was slightly longer in the high-dose group (5.27 days) when compared to the control (4.25 days). As the increase in the hihg-dose group mean value was slightly above a normal cycle length of 5 days, no test item-related effect is considered.

For a detailed description of the findings in tabular form, please refer to the attachment below (under Attached background material).

Effect levels

Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
62.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
bladder
kidney
ureter
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study and based on the test item-related finding, the NOAEL is determined to be 62.5 mg/kg bw/day for general toxicity.