Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 243-718-1 | CAS number: 20298-69-5
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
![](https://echa.europa.eu/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/print_toxicological-information.png)
Endpoint summary
Administrative data
Description of key information
Key study: NOAEL in 13 weeks sub-chronic study (according to OECD TG 408): >=423 mg /kg bw
Supporting study: NOAEL in 10 wks extended repeated dose / reproscreen study (extended OECD TG 422): >=437 mg/kg bw
Key value for chemical safety assessment
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- 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
- 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
- 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 - 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
- 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 - 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'
- 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
- 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
- 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
- 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.
Reference
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.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
- Study duration:
- subchronic
- Species:
- rat
Mode of Action Analysis / Human Relevance Framework
The key finding in rat after exposure to Verdox is the presence of thealpha 2u-globulin nephropathyin the kidneys of the male rat. This is kidney decrease typical for male rats (Swenberg, 1993). Alpha-2u-globulins can be found in the lysosomes of the cells. Hydrocarbon types of substances are processed in this cell organelle, possibly when the conjugation pathway is insufficient
(substance cannot be further processed in the mitochondrion for energy and the lysosomes take over from there). The lysosomes and mitochondrion are two cell organelles which show a close collaboration (Raimundo et al., 2016).
These hydrocarbons bind to these alpha 2 globulins and are transported to the kidneys. After releasing the substance into the urine the alpha2u globulins have a tendency to agglomerate in the renal tubulars and cause this alpha-hydrocarbon nephropathy, because the arrival of the protein is higher than the breakdown (Swenberg, 1993). Only male rat seem to develop this alpha-hydrocarbon nephropathy and therefore is considered a rat specific effect and not a human specific effect (Swenberg, 1993). Some efforts have been made to characterise substances causing thisalpha 2u-globulin nephropathy e.g. Melnick, 1992. These may be alcohols, ketones and possibly also acids. The key feature for initiation this binding to the alphy2u globulin is that the substance has a lipid (hydrocarbon) bulky backbone and at least one hydrogen donor to be able to bind to this proton or globulin. Therefore secondary and tertiary alcohols, ketones that can be reduced to alcohols are susceptible to binding (Borghoff et al., 1991).
Borghoff, S.J., Miller, A.B., Bowen, J.P. and Swenberg, J.A., 1991, Characteristics of chemical binding to alpha2u-globulin in vitro-Evaluation structure-activity relationships, Toxicol. Appl.Pharmacol. , 107, 228-238.
Melnick, 1993,Regulatory Toxicology and Pharmacology, An alternative hypothesis on the role of chemically induced protein droplet (α2u-globulin) nephropathy in renal carcinogenesis,Regulatory Toxicology and Pharmacology, 16,111-125,
Raimundo N1,Fernández-Mosquera L2,Yambire KF2,Diogo CV2, 2016, Mechanisms of communication between mitochondria and lysosomes,Int J Biochem Cell Biol.,79:345-349.
Swenberg, J.A., 1993, Alpha 2u-globulin nephropathy: review of the cellular and molecular mechanisms involved and their implications for human risk assessment,Environ Health Perspect., 101, Suppl. 6.,https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1520017/, site visited, June 2018
Additional information
Verdox dietary 90 -day study (OECD TG 408)
A sub-chronic (13-week) repeated-dose toxicity studywas 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.
Verdox extended >=10 wks OECD TG 422 study
In a dose-range finding study, rats received diets containing containing 0, 1155, 2310, 7700 and 15400 mg Verdox/kg diet (intented dose of 0, 75, 150, 500 and 1000 mg/kg body weight/day) for two weeks (4 rats/sex/dose). No treatment-related clinical signs were observed. Mean body weights in females from the mid- and top-dose groups were decreased. Body weight changes were reduced in males of the high- and top-dose groups and in female animals of the top-dose group. Food consumption was decreased in male and female animals of the high- and top-dose groups between 13 and 17%. These effects are expected to be due to the palatability of Verdox being a fragrance which may smell awkwardly for rats and therefore not tasty. At necropsy, kidneys of the male animals of all dosing groups showed a pale appearance. The relative weight of the livers of male and female animals of the top-dose group of 15400 mg/kg diet was statistically significantly increased up to 20 and 17%, respectively, which can be considered (close to or) at metabolic limit of saturation because the substance is expected to be fully absorbed based on its molecular weight and physico-chemical properties. At 7700 mg/kg diet the increases were 11 and 5%, respectively. Diet analysis showed that the diets were not stable at (animal) room temperature. Therefore, in the main study, the diets had to be refreshed daily. Based on the result of the current study and in consultation with the sponsor, the following dose levels were proposed for the subsequent main study: 0, 800, 2500 and 7500 mg Verdox/kg diet (corresponding with a nominal expected dose level of 0, 75, 200 and 500 mg/kg body weight) for the animals of the low-, mid- and high-dose groups, respectively. These level were expected no to affect the palatability of the diet and /or overloading metabolic saturation.
Main study: In the GLP compliant combined repeated dose and reproduction / developmental screening study, performed according to OECD guideline 422, Wistar rats were treated with the test substance (0, 75, 200, and 500 mg Verdox/kg bw/day, nominal expected dose levels) in the diet, during a premating period of 10 weeks and during mating (1 week), gestation and lactation until postnatal day 4.
Analysis of the test substance: The actual test substance intake ranged between 46-69 for males and 42-71 for the females of the low dose group. For the mid dose group the actual test substance concentration ranged from 141-209 for the males and 129-209 for the females. For the high dose group the actual test substance concentration ranged from 415-617 for the males and ranged between 381 and 672 for females.
Clinical observationsduring the premating, gestation and lactation period did not reveal any treatment-related changes in the animal’s appearance, general condition or behaviour.Neurobehavioural observationsand motor activity assessment did not indicate any neurotoxic potential of the test substance. No toxicological relevant effects on body weight and food consumption were observed in both sexes.
Haematology: No effects on red blood cell variables or clotting potential were observed.
Clinical biochemistry: A statistically significant increase in urea-concentration was observed in males of all dosing groups which might be related to the observed kidney effects (α2u-microglobulin nephropathy).
Organ effects: Increased relative liver weight (14%) was observed in high dose males, which was considered as an adaptive response to increased physiological demand. A dose related increase in relative kidney weight was observed in mid and high dose males, which was related to the observed α2u-microglobulin nephropathy. No effects on organ weights were observed in females. Effects observed on kidney weights and urea-concentrations were considered to be related to α2u-microglobulin nephropathy which was confirmed by immune-cytochemical staining of the α2u-microglobulin protein in the cortical tubular epithelial cells. This effect observed in rats is generally regarded as of no toxicological relevance for humans. Macrosopic and microscopic examination did not reveal any treatment-related effects.
Fertility: No treatment-related effects were observed on sperm-parameters (epidydimal sperm motility, sperm count and morphology and testicular sperm count). No treatment related effects were observed on pre-coital time, mating index, duration of gestation, pre- and postimplantation loss, number of corpora lutea, number of implantation sites and number of live pups on lactation.
Developmental toxicity: No treatment related clinical signs in pups, no effect on sex ratio in pups on day 1 and 4 of lactation, no effects on the mean pup body weight and body weight gain on lactation days 1 and 4 and no treatment-related macroscopic observations in pups that were stillborn or that died during lactation were observed.
In conclusion on repeated dose toxicity: The NOAEL for Verdox for males and females in this study was ≥ 437 mg/kg body weight in the diet (based on the lowest intake of the test substance in lactating females).
In conclusion on fertility and developmental toxicity: Based on the absence of effects on fertility parameters and developmental parameters, the NOAEL for Verdox for fertility and developmental toxicity in this study was ≥ 437 mg/kg body weight in the diet, based on the lowest intake in lactating females. A NOAEL of 500 mg/kg body weight in the diet is equivalent to an overall intake of at least 505 mg/kg body weight/day for males and at least 437 mg/kg body weight/day for females.
Justification for classification or non-classification
Based on the results of the sub-chronic repeated dose toxicity study in rats and the repeated dose reproscreen study, classification and labelling for repeated exposure is not warranted in accordance with EU CLP (EC No. 1272/2008 and its updates).
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
![ECHA](/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/echa_logo.png)