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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Oct 2016 to 10 Feb 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 21st September 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Triskelion B.V., Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
cis-2-tert-butylcyclohexyl acetate
EC Number:
243-718-1
EC Name:
cis-2-tert-butylcyclohexyl acetate
Cas Number:
20298-69-5
Molecular formula:
C12H22O2
IUPAC Name:
2-tert-butylcyclohexyl acetate
Test material form:
liquid
Details on test material:
Test material used is consistent with the substance identity as described in section 1.2 of IUCLID - company composition for the quality described under the common name Verdox.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han IGS (Crl:WI(Han))
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6 weeks at the start of the treatment period
- Weight at study initiation: at initiation of treatment the mean body weight per group was 162-163 g for males and 128-129 g for females
- Housing: The animals were kept in macrolon cages with wood shavings (Lignocel) as bedding material, and strips of paper (Enviro-dri) and a wooden block as environmental enrichment. They were housed in groups of five, separated by sex. On the day of FOB testing and motor activity assessment, the animals were temporarily kept singly in macrolon cages. During urine collection, animals were kept singly in stainless-steel metabolism cages
- Diet: From their arrival until the end of the study, the rats received a powdered, cereal-based rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England; batch 2372 during quarantine and 2619 after quarantine was raised). The diets were provided ad libitum in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage.
- Water: Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, ad libitum.
- Acclimation period: about 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 45 - 65
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 Oct 2016 To: 9 (males) and 10 (females) Feb 2017

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
The animals of the test groups were kept on experimental diets prepared by mixing VRF1 (FG) diet with the appropriate amounts of the substance using a mechanical blender. First, a premix was made by mixing the substance with VRF1 (FG) diet (batch 2619), to obtain a concentration 50.000 mg substance/kg diet. Subsequently the premix was mixed with frozen VRF1 (FG) diet (batch 2619) to obtain the test concentrations required. The experimental diets were prepared shortly before the start of the study and subsequently every ca. 4 or 5 weeks. After preparation, the experimental diets were divided into daily amounts of diets that were stored in plastic bags in a freezer (<-18°C). Each day, one bag per group was removed from the freezer to feed the animals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
From all three batches of diets prepared in the study, samples were taken and stored in a freezer (at ≤ -18 °C) until analysis. The test substance was assayed by UPLC-high-resolution-MS, which was validated to conform to the following criteria:
- Linearity: the correlation coefficient of the calibration curve should be ≥ 0.996.
- Recovery: the mean recovery of the test substance from diet should be between 85 % and 115 % at each of the dose levels of the study.
- Repeatability: the relative standard deviation in the percentage recovery should be less than 10 % at each of the dose levels of the study.
- Specificity: signals should be corrected in case the signal obtained for blank samples was ≥ 5 % of the signal obtained for low-dose samples.

The following analyses were conducted during the study:
- Homogeneity and content of the test substance at each dose level (5 samples of each test diet, taken at left top, right top, middle, left bottom and right bottom of the container, and 1 sample of the control diet were analyzed in duplicate) in one batch
- Content of the test substance at each dose level (one sample per dose level and one control sample) in two batches.
- Stability of the test substance under experimental conditions (one sample per dose level and one control sample of one batch, after storage for 1 day in the animal room and after storage for at least 5 weeks in the freezer (≤ -18 °C).
Duration of treatment / exposure:
13 weeks, 7 days per week (during the scheduled termination early in week 14, the animals were kept on their test diet until overnight fasting prior to necropsy).
Frequency of treatment:
continuously
Doses / concentrationsopen allclose all
Dose / conc.:
800 other: ppm; 80 mg/kg bw nominal
Remarks:
based on the average food consumption of control animals (14.9 g/day), and the average weight of control animals on day 42 of the study (281.9 g), the anticipated dose would be 42 mg/kg bw/day
Dose / conc.:
2 500 other: ppm; 250 mg/kg bw nominal
Remarks:
based on the average food consumption of control animals (14.9 g/day), and the average weight of control animals on day 42 of the study (281.9 g), the anticipated dose would be 132 mg/kg bw/day
Dose / conc.:
7 500 other: ppm; 750 mg/kg bw nominal
Remarks:
based on the average food consumption of control animals (14.9 g/day), and the average weight of control animals on day 42 of the study (281.9 g), the anticipated dose would be 396 mg/kg bw/day
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of a 14-day DRF for a previously performed 70-90-days extended OECD TG 422 study (Triskelion report V20063; A. Wolterbeek, 2012). In the DRF the dose of ca 15400 mg/kg diet (nominal 1000 mg/kg bw) was used. In this DRF at this high dose decrease in food consumption and body weight was seen and these effects were related to the palatability of the test item Verdox. Verdox is a fragrance which can smell awkwardly for rats and is therefore not tasty either. At this 15400 mg/kg diet in the DRF the relative liver weight increased >=17% in males and females, which we considered to be due to metabolic saturation. This liver weight increase is also expected in combination with Verdox’s MW (196) and physico-characteristics, which indicate full and fast oral absorption. See table below for effects during DRF
The dietary 80-90-day Repeated dose / Reproscreen study with the highest dose of 7500 mg/kg diet (converted to nominal 500 mg/kg bw) showed decrease in body weight gain (ca. 15%), which was considered test item related but not adverse. Based on this the 7500 mg/kg diet was sufficiently high to present (absence of adverse) effects for the OECD TG 408 study and not muddle the outcome of the study due to potential palatability issues. In this Repeated dose / Reproscreen study the relative liver weights in males were increased around 14%, which we related to increased metabolic capacity and considered this sufficiently high to set the maximum dose levels at 7500 mg/kg diet in the follow up OECD TG 408 study. See table below for effects in the extended OECD TG 422.

Dose selection according to the OECD TG 408 guideline:
In the OECD 408 guideline at section 14 it is presented that the highest dose level should induce toxicity but not death or severe suffering. The body weight (gain) effects found in the 14-day DRF at 15400 mg/kg diet (nominal 1000 mg/kg bw) were related to reduced food consumption. This reduction was considered due to palatability and therefore higher dietary doses were not used to limit suffering for the animals. Beside effects on body weight (gain) and food consumption the increase in liver weight in the 14-day DRF was around 10-5% at 7700 mg/kg diet (nominal 500 mg/kg bw), which we considered sufficiently close to a tolerable dose for the metabolic capacity of the liver. Mainly based on the expected palatability at higher doses, the 7500 mg/kg diet was selected as the highest dose.

Summary of key effects in DRF and extended OECD TG 422
DRF
Mg/kg diet Body weight Body weight gain Food consumption / per animal Relative liver weight
Males
1155 Not applicable Not applicable Not applicable Not applicable
2310 Not applicable Not applicable Not applicable Not applicable
7700 No effect -18% -17% +11%
15400 No effect -25% -17% +20%
Females
1155 Not applicable Not applicable Not applicable Not applicable
2310 Not applicable Not applicable Not applicable Not applicable
7700 -25% -13% +5%
15400 -6% -25% -15% +17%

Extended OECD TG 422
Mg/kg diet Body weight Body weight gain Food consumption / per animal Relative liver weight
Males
800 Not applicable Not applicable Not applicable Not applicable
2500 Not applicable Not applicable Not applicable Not applicable
7500 -7% -15% Not affected +14%
Females
800 Not applicable Not applicable Not applicable Not applicable
2500 Not applicable Not applicable Not applicable Not applicable
7500 No real effects -16% only during premat Not affected N No effects

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. All cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. All abnormalities, signs of ill health or reactions to treatment were recorded.

DETAILED CLINICAL OBSERVATIONS:
In addition to the above daily general clinical observations, detailed clinical examinations (in an arena outside the home cage) were performed on all rats prior to the first exposure and then once weekly throughout the study.

BODY WEIGHT:
The body weight of each animal was recorded once during the acclimatization period (day -3), at initiation of treatment (day 0), and once per week thereafter. The animals were weighed prior to scheduled necropsy in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was measured per cage by weighing the feeders. The consumption was measured daily throughout the treatment period for all animals in the cage. The results were expressed in g per animal per day.
- The intake of the test substance per kg body weight per day was calculated from the intended nominal dietary concentration of the test substance corrected for loss during the preparation of experimental diets and storage in the animal room, the weekly feed consumption and the mean of the body weight at the beginning and end of the pertaining week.

WATER CONSUMPTION:
Water consumption was measured per cage, by weighing the drinking bottles daily, during 5-day periods in weeks 1, 6 and 11. The results were expressed in g per animal per day.

OPHTHALMOSCOPIC EXAMINATION:
Ophthalmoscopic observations were made prior to the start of treatment (on day -7) in all rats, and in the last week of the treatment period in all rats of the control group (1) and the high dose group (4). Because no treatment-related ocular changes were observed in the high-dose group, eye examination was not extended to the animals of the intermediate-dose groups at the end of the study. Eye examination was carried out using an ophthalmo-scope after induction of mydriasis by a solution of atropine sulfate.

HAEMATOLOGY:
At necropsy, blood samples were taken from the abdominal aorta of all rats whilst under CO2/O2 anesthesia. The rats were fasted overnight before necropsy (water was freely available). EDTA or citrate (for prothrombin time) were used as anticoagulant. Blood samples were discarded after analysis. In each sample the following determinations were carried out: hemoglobin (Hb), packed cell volume (PCV), red blood cells (RBC), reticulocytes, total white blood cells (WBC), differential white blood cells (Lymphocytes, neutrophils, eosinophils, basophils and monocytes), prothrombin time and thrombocytes.
The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC).

CLINICAL CHEMISTRY:
At necropsy, blood samples were taken from the abdominal aorta of all rats whilst under CO2/O2 anesthesia. The rats were fasted overnight before necropsy (water was freely available). The blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. Plasma samples were discarded after analysis. The following measurements were made in the plasma: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin (calculated), urea, creatinine, (fasting) glucose, bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl) and inorganic phosphate (PO4).

URINALYSIS:
In week 13 male rats were deprived of water for 24 hours and of food during the last 16 hours of this period. During the last 16 hours of deprivation, the rats were individually kept in stainless-steel metabolism cages and urine was collected. Females were deprived of food and water according to the same schedule, with one exception; they inadvertently had access to water for 1 h after an initial 7 h water deprivation. Subsequently, they were deprived of food and water for 16 hours while they were housed in stainless-steel metabolism cages for collection of urine. All urine samples were discarded after analysis. The following determinations were carried out in individual samples: volume (to investigate the concentrating ability of the kidneys), density (to investigate the concentrating ability of the kidneys), appearance, pH, glucose, microscopy of the urinary sediment (red blood cells, white blood cells, epithelial cells, amorphous material, crystals, casts, bacteria, worm eggs, sperm cells), occult blood, ketones, protein, bilirubin and urobilinogen.

NEUROBEHAVIOURAL EXAMINATION:
Behavioral endpoints (Functional Observation Battery and motor activity assessment) were investigated in all rats at the end of the study (week 12).
Sacrifice and pathology:
GROSS PATHOLOGY:
Early in week 14, after overnight fasting (water was freely available), the animals were killed on two successive working days in such a sequence that the average time of killing was approximately the same for each group. The animals were killed by exsanguination from the abdominal aorta under CO2/O2 anesthesia and then examined grossly for pathological changes.
The following organs were weighed (paired organs together) as soon as possible after dissection to avoid drying, and the relative organ weights (g/kg body weight) were calculated on the basis of the terminal body weight of the animals: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles (with coagulating glands), spleen, testes, thymus, uterus.

HISTOPATHOLOGY:
The tissues to be examined microscopically were embedded in paraffin wax, sectioned and stained with hematoxylin and eosin.
Histopathological examination (by light microscopy) was performed on all tissues and organs listed below, of all animals of the control group (1) and the high-dose group (4) with one exception: kidneys from all animals (groups 1-4) were investigated for renal pathology. Because no treatment-related changes were observed in the high-dose group, histopathology for organs other than the kidney was not extended to the intermediate-dose groups. The following organs and tissues were examined: adrenals, aorta, axillary lymph nodes, brain (brain stem, cerebrum, cerebellum), cecum, colon, duodenum, epididymides, esophagus, eyes, GALT (gut associated lymphoid tissue, including Peyer's patches), heart, ileum, jejunum, kidneys, liver, lungs, mammary gland (females), mesenteric lymph nodes, nerve-peripheral (sciatic), ovaries, oviducts (=fallopian tubes), pancreas, parathyroid, parotid salivary glands, pituitary, prostate, rectum, seminal vesicles+ coagulating glands, skeletal muscle (thigh), skin (flank), spinal cord (retained in vertebral column, at least three levels were examined microscopically (cervical, mid-thoracic and lumbar)), spleen, sternum with bone marrow, stomach (non glandular (‘forestomach’) and glandular (fundus, pylorus) parts were examined microscopically), sublingual salivary glands, submaxillary salivary glands, testes, thymus, thyroid, trachea/bronchi, urinary bladder, uterus (with cervix), vagina, all gross lesions.
Other examinations:
Renal α2-urinary globulin was assessed by immunohistochemistry. Sections of the kidneys of all male animals (groups 1-4) were processed for immunohistochemical staining of α2-urinary globulin using a mouse-anti-rat α2-urinary globulin antibody (lot nr. R12273504A, SSI, Copenhagen, Denmark).
Statistics:
Please refer to 'other information on materials and methods incl. tables'

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Tail kink was noted in one animal in the control group. Sparsely haired area(s), skin wounds and/or skin encrustations were found for part of the exposure periode in three animals in the control group, five animals in the low dose group, one animal in the middle dose group and three animals in the high dose group. These were normal findings and occurred randomly among the groups. Thus, there were no treatment-related clinical signs.
An overview of the clinical signs can be found in tabular form in the attached study report on page 33.
Mortality:
no mortality observed
Description (incidence):
None of the rats died during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights were slightly (within 10%) decreased in the high-dose group. The differences with the controls were statistically significant in high-dose males at all stages throughout the study and in high-dose females at most stages from day 21. Body weighs were not affected in the low-and mid-dose groups.
An overview of mean body weight can be found in tabular form in the attached study report on page 35 to 38.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- During the first two days of the study, food intake was reduced in the mid- and or the high-dose group in both sexes. Thereafter, food intake tended to be decreased in males of the high-dose group (on average, food consumption was decreased by 13% compared to control animals) but the differences with the controls were not always consistent.
- Due to the decreased food intake per kg body weight with increasing age of the rats, the intake of the test substance per kg body weight gradually decreased in all groups. The overall mean intake of the test substance in the low-, mid- and high-dose group was 47, 147 and 450 mg/kg body weight/day for males, and 55, 166 and 486 mg/kg body weight/day for females, respectively. These levels are based on the nominal levels of the test substance in the diets, and are not yet compensated for the actual levels achieved (see any other information on results incl. tables). If the achieved levels are taken into consideration the overall intake was 37, 118 and 423 mg/kg body weight/day for males, and 43, 133 and 457 mg/kg body weight/day for females, respectively.
An overview of food consumption and test substance intake can be found in tabular form in the attached study report on page 39 to 54 and 55 to 58 respectively.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
During the first day of exposure only (day 0-1), water consumption was lower in the high dose group (males and females) and the mid dose group (females only). The reduction in water consumption was slight and transient, therefore it is not considered adverse. In week 11, slightly increased water consumption was observed in males from the high-dose group. Otherwise, no relevant differences in water consumption were noted among the groups.
An overview of water consumption can be found in tabular form in the attached study report on page 59 to 62.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopy did not reveal any treatment-related changes.
An overview of ophthalmological findings can be found in tabular form in the attached study report on page 34.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related effect was found on red blood cell/coagulation or white blood cell counts. A slight, though statistically significant increase in the percentage of lymphocytes was found in females from the high dose group. This finding was not reflected in significant changes in the absolute lymphocyte count and is considered a chance finding.
An overview of haematological findings can be found in tabular form in the attached study report on page 63 to 66.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significantly increased alkaline phosphatase concentration and decreased plasma glucose concentration was observed in males from the low dose group. In female animals from the high dose group, a slight but statistically significant decrease in chloride concentration was found. These are considered chance findings and are not considered to be toxicologically relevant.
An overview of clinical biochemistry findings can be found in tabular form in the attached study report on page 67 to 70.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In high-dose males, the urinary volume was statistically significantly increased and specific gravity was decreased. Such finding might point to impaired concentrating ability of the kidneys. However, the changes were only very slight and rather related to the somewhat higher water intake of high-dose males at the end of the study. Similar phenomena (statistically significantly decreased specific gravity associated with a somewhat higher urinary volume) were noted in low-dose females without confirmation at higher dose levels. Therefore no toxicological significance was attached to these findings.Semi-quantitative (dipstick) measurements did not reveal relevant changes. A statistically significant increase in occult blood was noted in high-dose males, whereas this parameter was decreased in females of the mid- and high-dose groups. The urinary glucose content was slightly increased in three females of the high-dose group, but similar findings were noted in males of all groups, including the controls. Other statistical significant changes noted were decreased protein in low-dose females and decreased bilirubin in females of all dose groups. These findings are not toxicologically relevant and are considered chance findings. Microscopy of the urinary sediment showed a statistically significantly increased amount of epithelial cells in the urinary sediment in low- and high-dose males. In mid-dose males this finding was less evident. A decreased amount of amorphous material in high-dose females was considered chance finding.
An overview of urinalysis findings can be found in tabular form in the attached study report on page 71 to 76.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The results of the neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the substance in rats
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant differences were observed in organ weight between exposed groups and controls:
- The relative weight of the kidneys was increased in males of the mid- and high-dose group (12% and 24%, respectively).
- The relative weight of the liver was increased in high-dose males (12%).
- The absolute and relative weights of the uterus were increased in high-dose females (absolute: 72%, relative: 77%).
- In high-dose males, the relative weights of the brain (9%) and testes (10%) were increased (the latter finding was not statistically significant). These changes are ascribed to growth retardation. During growth retardation, rats tend to maintain their (absolute) weights of brain and testes, and there is a well-known inverse correlation between terminal body weight and the relative weight of these organs.
- An incidental decrease in relative heart weight (-7%) was noted in low-dose males.
An overview of organ weight findings can be found in tabular form in the attached study report on page 77 to 80.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy revealed no treatment-related abnormalities. The findings were considered unremarkable and part of the background pathology of rats of this strain and age.
An overview of gross pathological findings can be found in tabular form in the attached study report on page 81.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Microscopic examination revealed a dose-dependent increase of hyaline droplets accumulation in the proximal tubular epithelium cells in the kidneys of the male animals. In the low-dose group, six out of ten animals showed minimal to mild hyaline droplet accumulation. In the mid-dose group, all ten animals showed minimal to mild hyaline droplet accumulation. In the high-dose group, all ten animals showed mild to moderate hyaline droplet accumulation.
- A dose-dependent increase of basophilic tubuli in the kidneys of the male animals was observed. In the low-dose group, six out of ten animals showed minimal to mild basophilic tubuli. In the mid-dose group, all ten animals showed minimal to mild basophilic tubuli. In the high dose group all ten animals showed mild to moderate basophilic tubuli.
- A dose-dependent increase was observed of dilated tubuli, filled with eosinophilic debris (Granular casts), in the kidneys of the male animals. In the low-dose group three out of ten animals showed minimal to mild granular casts. In the mid-dose group, nine out of ten animals showed minimal to mild granular casts. In the high-dose group, nine out of ten animals showed mild to moderate granular casts.
- The above histological observations are in agreement with the description of nephrotoxicity induced by the accumulation of α2-urinary globulin in males, characterized by enlarged lysozomes, granular casts (as a result of the exfoliation of the proximal tubule epithelium) and tubular cell proliferation (basophilic tubules) (Swenberg, 1993).
To confirm the presence of α2-urinary globulin, we have performed immunohistochemistry with a specific antibody against α2-urinary globulin. The results showed that the granular casts were positive for α2-urinary globulin staining, indicative of α2-urinary globulin accumulation in the exfoliated tubule cells. In addition, the hyaline droplets were also positive for α2-urinary globulin staining.
- The remaining histopathological findings were considered unremarkable and part of the background pathology of rats of this strain and age.
An overview of histopathological findings can be found in tabular form in the attached study report on page 82 to 87.
Histopathological findings: neoplastic:
no effects observed

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
>= 423 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Absence of human relevant adverse effects at the highest dose tested
Remarks on result:
other: no adverse effects of human relevance observed in male rats either
Dose descriptor:
LOAEL
Effect level:
37 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other: effect not relevant for human

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
37 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Any other information on results incl. tables

Analysis of the experimental diets

- The analysis method for the substance was successfully validated, all pre-determined validation criteria were met.

- Comparison of test substance concentration in diet samples taken from various locations within the mixing chamber showed that the test substance was homogeneously distributed in the diets.

- The test substance in diet was considered to be stable when stored in the freezer (at < -18 °C) in a closed container for 10 weeks (the relative decrease of substance concentration was less than 10% for all dose levels). When stored at ambient temperature in an animal room in an open container for one day, the test substance concentration decreased to 89.6%, 86.3% and 88.1% of the nominal concentration for the low-, mid- and high-dose groups respectively; because this was just below the lower limit of 90% for all dose groups, a slight loss of test substance from the diets was assumed during storage in the animal room during one day.

- The content of the test substance in diet was considered to be “close to intended” if the mean measured concentration did not deviate more than 10% from the intended concentration. The mean concentrations of the test substance in the test diets showed some variation (Table 1).

Table 1: Levels of substance detected in the test diets.

Intended concentration of the substance in diet

Levels found (relative difference compared to intended)

8

November 2016

9

December 2017

6 January 2017

Average

800 mg/kg

-15%

-12%

-25%

-17%

2500 mg/kg

-15%

-6%

-22%

-14%

7500 mg/kg

-4.5%

-6%

-2.5%

-4%

Test substance concentrations in the low- and mid-dose diets did not meet the criterion “close to intended” (between 90% and 110% of the intended concentration). The fluctuation in the above figures is ascribed to evaporation of the test substance from the diet-test substance mixture during preparation. Therefore it is concluded that the intended concentrations were achieved for the high-dose diet, not for the low- and mid- dose diets.

 

Recalculation of test substance concentration in diet:

Because in the low- and mid-dose group the intended concentrations were not met and because there was a slight loss of test substance from the diet upon use in the animal room during one day, the achieved test substance concentration in diet was recalculated. Assuming linear degradation/evaporation of the test substance from the experimental diet, the average loss in the animal room was assumed to be half the measured loss in 24h. The corrected results (achieved concentrations) are presented in the table below:

Intended concentration of the substancein diet

Loss during preparation

(average of three analyses)

Loss after storage in the animal room for 24 hours

Assumed average loss in the animal room

Achieved concentration of Verdox in diet

800 mg/kg

-17%

-10%

-5%

629 mg/kg

2500 mg/kg

-14%

-14%

-7%

2003 mg/kg

7500 mg/kg

Close to intended

-12%

-6%

7054 mg/kg

These concentrations have resulted in the following doses:

 

Intended concentration of the substance in diet

Achieved dose, males (mg/kg b.w./day)

Achieved dose, females (mg/kg b.w./day)

800 mg/kg

37

43

2500 mg/kg

118

133

7500 mg/kg

423

457

Overall conclusion:

The rats were exposed to concentrations that were slightly below the intended concentrations. Therefore, the achieved concentrations (instead of the intended concentrations) are used as NOAEL/LOAEL in the conclusions of the current study.

Applicant's summary and conclusion

Conclusions:
Oral administration of the substance in diet, for a period of 13 weeks to Wister Han IGS (Crl:WI(Han)) rats revealed a NOAEL >=423 mg/kg bw. It can be a LOAEL of 37 mg/kg bw/ day for male rats based on on the observed α2-urinary globulin-mediated nephropathy but is not relevant for human.
Executive summary:

A sub-chronic (13-week) repeated-dose toxicity study was performed according to OECD TG 408 and GLP principles (2017). Animals were given intended concentrations of 800, 2500 and 7500 mg/kg in diet based on the dose range finder (DRF) for a >=10 weeks Repeated dose / Reproscreen study. In the DRF study the palatability of the substance at >= 7700 mg/kg diet (nominal 500 mg/kg and 1000 mg/kg bw) was influencing the body weight (gain) and food consumption. Based on these effects the maximum dose was set to 7500 mg/kg diet. In addition, significant and dose related increased relative liver weights were seen at the 7700 and 15400 mg/kg diet (increase at least of 11 and 5% for males and females, respectively at 7700 and 20 and 17%, for males and females, respectively, at 15400 mg/kg diet. This relative liver weight increase indicates that metabolic capacity is increased close to the maximum non-adverse level at 7500 mg/kg diet.

Analysis of the diets: Analysis for the actual concentration of the test substance in the experimental diets showed some loss of the test substance during preparation and/ or during storage for 24 hours in the animal room. Therefore, the test substance concentrations in the test diets were corrected. The achieved dietary concentrations were 629, 2003 and 7054 mg/kg diet for the low-dose, mid-dose and high-dose groups, respectively. Compensated for the actual levels achieved, the overall intake was 37, 118 and 423 mg/kg body weight/day for males, and 43, 133 and 457 mg/kg body weight/day for females, respectively.

Clinical signs: The administration of the test substance was well tolerated at all dose levels, and did not induce any relevant changes in general condition, feed or water intake, neurobehavioral observations, ophthalmoscopy, hematology, clinical chemistry, urinalysis, or in macroscopic examination at necropsy.

Body weightswere decreased in the high-dose group in both sexes, but the differences with the control group were only slight (within 10%).

Heamatology: No adverse effects were seen.

Clinical biochemistry: No adverse effects were seen.

Organ effects: The relative weight of the liver was slightly increased (12%) in high-dose males. This finding was not accompanied by pathology and is anticipated as an adaptive response. Furthermore, the relative weight of the kidneys was increased in males of the mid- and high-dose group (12% and 24%, respectively). Renal pathology was observed in male rats of all dose groups, but not in females. Immunohistochemical staining forα2-urinary globulin showed that the observed pathological changes were mediated byα2-urinary globulin which is specific for male rats. The incidence and/or severity of the effects aggravated with increasing dose levels and the findings were already present in low-dose males. These findings were accompanied by an increase in the relative weight of the kidneys in male animals of the mid-dose and high-dose group and by an increase of epithelial cells in the urinary sediment in low- and high-dose males, which resulted in increased kidney weight. It should be noted thatα2-urinary globulin-mediated renal pathology, observed in this study at 37 mg/kg bw (629 ppm and a LOAEL for male rats) is not considered relevant for humans and that no other major effects were noted in this study. Therefore, the human relevant NOAEL should be set at >=7054 mg/kg diet, corresponding to a test substance intake of >=423 mg /kg body weight/day based on the absence of human relevant effects at the highest dose tested in female rats. This value is additional supported by the absence of human relevant effects in male rats at the highest dose tested. The absolute and relative weights of the uterus were increased in high-dose females (up to 77%). In relation, a relatively high number of females in the high-dose group macroscopically showed a swollen uterus (2 controls versus 7 high-dose rats) which is ascribed to random variation in the estrous stage. Therefore the increase in uterus weight is considered a chance finding. This is further supported by the absence of any relevant histopathology.

Conclusion: No human relevant adverse effects were seen >=423 mg/kg bw.