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EC number: 227-575-2 | CAS number: 5894-60-0
- 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
Toxicity to reproduction
Administrative data
- Endpoint:
- extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity
Cross-reference
- Reason / purpose for cross-reference:
- data waiving: supporting information
Reference
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 07 Dec 2020 - 15 Apr 2021
- 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:
- 25 June 2018
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- other: Wistar, Crl: WI(Han)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: 217 – 251 g (males); 118 – 150 g (females)
- Housing: animals were kept in groups of 5 animals/sex/group/cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet for rats and mice (Altromin Spezialfutter GmbH & Co. KG, Lage, Germany), ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals), ad libitum
- Acclimation period: at least five days under laboratory conditions
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12 - Route of administration:
- oral: gavage
- Vehicle:
- paraffin oil
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Based on the results of stability testing (Eurofins Munich Study No. STUGC20AA2272-1), the test item formulations were prepared within the determined stability time frame. The prepared formulation was stored protected from light and at room temperature.
The test item was weighed into a tared container, which has been proven to guarantee compatibility with the test item and test item stability for the duration of storage on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulation was alternately vortexed and/or stirred until visual homogeneity was achieved.
After homogenization the formulation was overlaid with argon to prevent instability caused by repeated contact of the test item formulation with air.
VEHICLE
- Justification for use and choice of vehicle: the vehicle was selected based on the test item’s characteristics
- Concentration in vehicle: 10, 25 and 62.5 mg/mL
- Amount of vehicle: 4 mL/kg bw/day
- Lot/batch no.: 0000855509, 0001849607, 0001929619 - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Analysis of test item formulations was performed at the Analytical Department of the Test Facility using a validated GC-FID (Gas Chromatography – Flame Ionization Detector) method.
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
As the test item formulation was shown to be homogenous in the preceding GLP analytical validation study (after 30 min without stirring), samples were not collected during the study for the investigation of homogeneity and only samples were taken for substance concentration in study Week 1, 5, 9 and in the last week of treatment for all doses (16 samples in total).
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 3 mL). The A-samples were analysed at the test facility and until then stored under appropriate conditions based on available stability data. The B-samples were retained at below - 15 °C at the test facility and discarded after completion of the final study report.
Concentration analysis of formulation samples was determined at three concentrations, 10 mg/mL, 25 mg/mL and 62.5 mg/mL in study Weeks 1, 5, 9 and in the last week of the study. The mean recoveries observed for the low dose (LD) group was between 88.6% and 106.5% of the nominal value, between 84.5% and 105.8% of the nominal value for the medium dose (MD) group and between 84.8% and 104.6% of the nominal value for high dose (HD) group. The mean recoveries observed in the LD, MD and HD groups were 101.5%, 98.4%, and 96.1% of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 15% based on rounded values.
However, sample analysis of Week 5 had to be repeated two times as MD and HD values were out of acceptance criteria. The sample preparation was adjusted to reduce overall preparation time and minimize contact to air during preparation steps. Thereafter, sample concentration was within acceptance criteria. - Duration of treatment / exposure:
- 90 days (control and test groups)
90 days and 28 days post-exposure observation period (satellite control and high-dose group) - Frequency of treatment:
- once daily, 7 days/week
- Dose / conc.:
- 40 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 250 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 10 (main study)
5 (satellite control and high-dose groups) - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
In the dose range finding study, two male and two female Wistar rats were treated with test substance at dose levels of 40, 80, 120, 200 or 300 mg/kg bw/day over a period of 14 days. There was no test item-related effect observed on mortality, clinical signs, body weight, food consumption or haematology. At necropsy, an abnormally coloured jejunum and ileum was found in one male dosed with 200 mg/kg bw/day. The thymus was enlarged in both males dosed with 200 mg/kg bw/day and in both females each dosed with 200 mg/kg bw/day or 300 mg/kg bw/day. In one male dosed with 300 mg/kg bw/day grey foci in the stomach fundic region were observed correlating with inflammatory lesions found in the histopathological examination. Histologically, test item-related findings consisted of inflammatory and degenerative lesions in the stomach including squamous cell hyperplasia in the forestomach in one male at 120 mg/kg bw/day and in both sexes at all higher doses. The finding was associated with forestomach submucosa inflammation (both males and one female), inflammation of the muscularis of the glandular stomach (one male only) and forestomach ulceration (one male only) at 300 mg/kg bw/day.
The highest dose level for the main study was chosen with the aim of inducing toxic effects, but not death or severe suffering including severe effects like ulceration and necrosis. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.
- Fasting period before blood sampling for clinical biochemistry:
yes, animals were fasted overnight.
- Rationale for selecting satellite groups: in order to allow a detection of possible delayed occurrence or persistence of or recovery from toxic effects.
- Post-exposure recovery period in satellite groups: 4 weeks - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
general clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
DETAILED CLINICAL OBSERVATIONS: Yes
- Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge) and piloerection were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.
BODY WEIGHT: Yes
- Time schedule for examinations:
the body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment and recovery period.
FOOD CONSUMPTION: Yes
- Food consumption was measured weekly during the treatment and recovery period.
OPHTHALMOSCOPIC EXAMINATION: Yes
- Ophthalmological examinations, using an ophthalmoscope were made on all animals before the first administration and in the last week of the treatment period as well as at the end of the recovery period in the recovery animals.
HAEMATOLOGY: Yes
- Time schedule for collection of blood:
haematological parameters were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
- Anaesthetic used for blood collection: Yes
(ketamine / xylazin)
- Animals fasted: Yes
- How many animals:
all animals
- Parameters checked in Table No. 1 were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
clinical biochemistry was examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in Table No. 2 were examined.
PLASMA/SERUM HORMONES: Yes
- Time of blood sample collection: end of the treatment and recovery period
- Animals fasted: Yes
- How many animals:
all animals
- Parameters checked in Table No. 3 were examined.
URINALYSIS: Yes
- Time schedule for collection of urine:
urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals.
- Parameters checked in Table No. 4 were examined.
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
once before the first exposure and once towards the end of the exposure period but not earlier than in Week 11, as well as in the last week of the recovery period multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests.
- Dose groups that were examined:
all animals - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes, one day after the last administration (study Day 91) all surviving animals of the treatment period and 4 weeks after the last administration all surviving animals of the recovery period (study Day 119) were sacrificed using anaesthesia (ketamine/ xylazin) and subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. All animals found dead and/or intercurrently euthanised for animal welfare reasons were subjected to a gross necropsy and the organs preserved for a histopathological examination.
Special attention was given to the gastrointestinal tract in order to assess possible corrosive effects, i.e.:
- The oral cavity was inspected for possible lesions (special attention was paid to the pharynx).
- The oesophagus was opened by a longitudinal cut with a scissor (without rinsing).
- The stomach was rinsed after opening with a cut and the mucosa was inspected.
- The small intestine was separated from the large intestine by a cut at the entry of the ileum into the cecum. Thereafter, the small intestine was cut into 3 pieces whereby the proximal end was marked by a shaped cut, and the intestine was gently rinsed with formalin (if the intestine was too full, small cuts allowed the outflow of ingesta and rinsing fluid).
- The cecum was opened by a longitudinal cut and rinsed. Colon and rectum was rinsed.
- All intestinal tissues were preserved.
ORGAN WEIGHTS: Yes, the wet weight of the organs (Table 5) of all sacrificed animals was recorded as soon as possible. Paired organs were weighed together. Weight of thyroid/parathyroid glands were measured after fixation. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded.
HISTOPATHOLOGY: Yes (Table 6), the tissues from all animals were preserved in 4% neutral-buffered formaldehyde except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol. Organs were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining for the animals of the control and high dose groups sacrificed at the end of the treatment period and any animal found dead or euthanised before the planned day of sacrifice.
For testis, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides.
For organs and tissues showing treatment-related changes in the high dose group, these examinations were extended to animals of all other dosage groups as well as to animals subjected to necropsy at the end of the recovery period.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discolouration possibly due to the test item was evaluated in the organs of all dose groups. - Optional endpoint(s):
- Examination of Fertility Parameters:
Vaginal smears were examined on the day of necropsy to determine the stage of oestrous cycle in all female animals of the main and recovery group.
At necropsy (one day after the last administration) and at the end of the recovery period, left epididymis and left testis of Group 1 and 4 males were separated and used for evaluation of sperm parameters (main and recovery). Epididymal sperm motility and testicular sperm count were evaluated in all male animals.
Sperm morphology slides were prepared from all male animals of Groups 1 and 4 (main and recovery). - Statistics:
- A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. Furthermore, statistical comparisons of data acquired during the recovery period was performed with a Student’s t-Test or Mann-Whitney U-Test when appropriate. These statistics were performed with Ascentos 1.3.4 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Slight to severe salivation was noted in 4/10 males and 5/10 females of the 40 mg/kg bw/day (LD) group, 9/10 males and 9/10 females of the 100 mg/kg bw/day (MD) group and in 15/15 males and 15/15 females of the 250 mg/kg bw/day (HD) group. Furthermore, moving the bedding was noted in 8/10 males and 6/10 females of the LD group, in 10/10 males and 9/10 females of the MD group and in 15/15 males and in 15/15 females of the HD group.
These clinical signs of moving the bedding and salivation were observed immediately after the dose administration and therefore were considered to be signs of a local reaction. This is a typical sign of irritation due to the test item rather than a systemic adverse effect, which was deemed to have no toxicological relevance.
Other clinical findings such as eyes closed, vocalization, piloerection, abnormal breathing, nasal discharge, coughing and sneezing, hunched posture, tremor were seen in single male and female animals of all dose groups and the control group.
Decedent animals showed clinical signs such as hunched posture, eyes closed, nasal discharge, abnormal breathing, dehydration, hairless area, increased salivation and moving the bedding.
Clinical symptoms like eyes closed, vocalization, piloerection, abnormal breathing, nasal discharge, coughing and sneezing, hunched posture, tremor were observed mostly transiently or on single days and within the normal background frequency. These symptoms are not considered to be test item-related.
On the first day of the recovery period, some males and females in the HD group were still moving the bedding and had increased salivation. No clinical signs were observed during the remainder of the recovery period.
No statistically significant effects on detailed clinical examination parameters were noted. - Mortality:
- mortality observed, treatment-related
- Description (incidence):
- One male of the HD group was found in moribund condition on study Day 50. The animal had abnormal breathing, nasal discharge, a hunched posture, severe piloerection, both eyes closed and was dehydrated. The major lesion of the latter animal was a lung that did not collapse due to moderate interstitial inflammation and slight accumulation of alveolar macrophages. This lesion was deemed to be due to accidental aspiration.
One female of the HD recovery group was found dead on study Day 86. The animal had moderately increased salivation and was moving the bedding on the days prior to its death. The death of this animal was most likely due to stomach ulceration.
No mortalities occurred during the recovery phase. - Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The test item had no effect on body weight development in the study. Overall the mean body weight increased normally during the treatment and recovery period in control as well as in dose group animals except a statistically significant lower body weight change in males in the MD and HD groups between study days 36 and 43.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- The test item had no toxicological relevant effect on food consumption in this study. Mean daily food intake of male and female animals was in the normal range of variation throughout the treatment and recovery period of this study and without considerable differences between the groups.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- Ophthalmoscopy examination did not reveal any test item-related effects.
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- A statistically significant increase of white blood cell count (WBC) in the HD females (~34% above control) and in platelet count (PLT) in LD and HD females was observed when compared to controls (~20% and ~25% above control, respectively).
At the end of the recovery period, white blood cell count (WBC), mean corpuscular volume (MCV), reticulocytes (RET), lymphocytes (LYM) and large unstained cells (LUC) were statistically significantly increased in males of the HD group when compared to the control group (~69%, ~4%, ~41%, ~14%, 100% above control, respectively) and mean corpuscular haemoglobin concentration (MCHC) and neutrophils (NEUT) were statistically significantly decreased in males of the HD group when compared to the control group (~4% and ~40% below control, respectively). Basophils (BASO) were statistically significantly decreased in females of HD group when compared to control (~63% below control).
At the end of the treatment period the prothrombin time (PT) was slightly but statistically significantly elevated in males and females of the HD group when compared to the control group (~10% and ~9% above control, respectively). After the recovery period, no statistically significant changes were recorded in blood coagulation.
As the respective values were in the range of historical control data, these changes are not deemed to be toxicologically relevant, but rather consistent with the background variability observed within this laboratory. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- At the end of the treatment period, a statistically significant decrease in aspartate-aminotransferase (ASAT) was found in HD males compared to controls (~29% below control) and a statistically significant increase in total bilirubin (TBIL) in MD males and total bile acids (TBA) in MD and HD males was observed when compared to controls (~13 %, ~117% and ~177% above control, respectively). In females, a statistically significant change was measured for alanine aminotransferase (ALAT) in the LD and HD groups (~18% and ~22% below control), in total protein (TP) in the HD group (~8 % below control) and in triglycerides (TG) in the LD group (~62% above control). The changes in biomarkers for liver toxicity did not correlate with any histopathological findings and were therefore assumed not to be adverse.
At the end of the recovery period, statistically significant changes were only noted for TP and albumin in the females of the HD group when compared to the control group (~7% and ~9% below control, respectively).
As the mentioned statistical significances at the end of the recovery period occurred only in one gender and all measured values were within the historical control data, a toxicological effect of the test item at the end of treatment or the recovery period could not be detected. - Endocrine findings:
- not specified
- Description (incidence and severity):
- The following ED-related parameters were investigated in the study: T3, T4 and TSH, cervix histopathology, coagulating gland histopathology, epididymis histopathology, epididymis weight, oestrus cyclicity, liver weight, mammary gland histopathology (males and females), ovary histopathology, ovary weight, prostate histopathology, prostate weight, seminal vesicles histopathology, seminal vesicles weight, sperm morphology, sperm motility, sperm count, testis histopathology, testis weight, thyroid histopathology, thyroid weight, uterus histopathology, uterus weight, vagina histopathology, vaginal smears, adrenals histopathology, adrenals weight, brain weight, pituitary histopathology and pituitary weight. For details, please refer to the respective result fields and the endpoint summary.
- Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- The urinalysis of animals sacrificed at the end of the treatment and the recovery period revealed no test item-related effects and all urinary parameters were in the normal range of variation.
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- In both males and females, no toxicological relevant effects were observed in any of the parameters of the functional observational battery before and at the end of the treatment or recovery period when compared to controls.
Before the start of the treatment (Week -1) statistically significantly more females in the MD group were asleep and consequently not moving in the cage when compared to animals in the control group. At the end of the treatment period statistically significantly more males in the LD and HD groups and females in the LD, MD and HD groups were asleep and consequently not moving in the cage compared to animals in the control group. In addition, statistically significantly more males in the HD group were grooming compared to animals in the control group and the body temperature of females in the LD, MD and HD groups was slightly but statistically significantly lower compared to the control group. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- At the end of the treatment period, mean absolute weight as well as mean organ to brain weight of the spleen and heart were statistically significantly reduced in males of the MD group when compared to the controls (~11%, ~11%, ~11% and ~11% below control, respectively). Mean organ to body weight ratio of the heart was statistically significantly reduced in females of the HD group when compared to controls (~7% below control).
At the end of the recovery period, mean absolute weight as well as mean organ to body and brain weight ratios of testes, pituitary gland and adrenal glands of HD males were statistically significantly higher when compared to control (~15%, ~11%, ~14%, ~48%, ~41%, ~46%, ~25%, ~21% and ~24% above control, respectively).
These changes in organ weight and organ to body and brain weight ratios did not correlate with any histopathological findings and are therefore assumed to not be adverse. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- No macroscopic changes that could be due to toxicologically relevant systemic effects of the test item were observed in any of the animals examined in this study.
The gross findings recorded in the survivors were within the range of normal background changes which may be observed in rats of this strain and age, or were with incidental gross appearances without corresponding histological changes or were with macroscopic alterations representing normal physiology.
Macroscopic examination of the decedent animals at necropsy listed in the HD group male a lung that failed to collapse, a gas filled stomach, duodenum, ileum, cecum and colon, a spotted abnormally coloured thymus and in the HD recovery group female a marble coloured lung, a gas filled stomach, jejunum and ileum, a stomach that had a spotted wall, dark red ileum and jejunum, dark red ovaries, a spotted abnormally coloured thymus, yellow mesenteric lymph nodes and autolytic eyes. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Gastrointestinal tract (Table 7):
Under the conditions of this study, one recovery female at 250 mg/kg bw/day died spontaneously during the course of study most likely due to stomach ulceration. One main test male at 250 mg/kg bw/day was sacrificed on study day 50 due to poor condition that was deemed to be due to accidental aspiration. Gross lesions were not remarkable. In contrast, histologically, there were primary findings indicative of local toxicity in the stomach at 250 mg/kg bw/day.
At 250 mg/kg bw/day, there was a number of inflammatory and degenerative findings in the stomach. They consisted of an increased focal to multifocal hyperkeratosis of the forestomach epithelium and inflammation in the forestomach submucosa and hyperplasia of the forestomach squamous epithelium. In a few cases, there was erosion and/or ulceration of the forestomach mucosa. Inflammation was also noted in one female at 100 mg/kg bw/day. In this dose, there was also a high incidence of squamous metaplasia at a minor degree of severity. After the recovery period, findings partially resolved.
Lungs (Table 8):
Findings due to study-related procedures and the vehicle used were noted in lungs.
Alveolar macrophages and interstitial inflammation were noted at a higher incidence in control animals compared to animals at 250 mg/kg bw/day. In a few animals, there was a minimal alveolar/bronchiolar hyperplasia. In addition, in one male at 250 mg/kg bw/day there was a small granuloma with multinuclear foreign giant cells.
All these lesions were deemed to be a consequence of accidental aspiration and related to the vehicle, paraffin oil. The reason for the higher incidence of findings in the control animals may be considered due to a higher viscosity of the vehicle compared to a mixture with test item.
In one female at 100 mg/kg bw/day, there was chronic pleural inflammation likely induced by a gavage accident.
Mesenteric lymph nodes (Table 9):
Findings due to study-related procedures and the vehicle used were noted in mesenteric lymph nodes.
In mesenteric lymph nodes, minimal to moderate accumulated macrophages containing an amorphous material were noted in all groups, including controls. The findings were noted at a slightly higher incidence and/or severity in animals at 250 mg/kg bw/day compared to other groups. After the recovery period, the findings were still present in males and females of the control group and at 250 mg/kg bw/day, with slightly higher severity in females at 250 mg/kg bw/day compared to control females.
In the decedent HD recovery group females, there were in addition moderate foamy histiocytes.
The findings in the mesenteric lymph nodes were deemed to be related to the paraffin oil effect: as indicated by several studies in rats, mineral hydrocarbons can induce accumulation of hydrocarbons as well as histiocytosis in the mesenteric lymph nodes (e.g. Firriolo et al. 1995 and Smith et al. 1996*). However, in this instance, the effects were slightly enhanced by mixture with the test item.
Other findings:
Indicators of stress were noted as hypertrophy in the adrenal cortices of a few control females and one case of minimal bone marrow atrophy in a control male.
The type, location and distribution of the remainder of findings did not distinguish controls from test item-treated animals.
References (*)
Firriolo, Janet M.; Morris, C. Fred; Trimmer, Gary W.; Twitty, Linda D.; Smith, Jacqueline H.; Freeman, James J. (1995): Comparative 90-Day Feeding Study with Low-Viscosity White Mineral Oil in Fischer-344 and Sprague-Dawley-Derived CRL:CD Rats. In: Toxicologic pathology 23 (1), S. 26–33. DOI: 10.1177/019262339502300104
Smith, J. H.; Mallett, A. K.; Priston, R. A.; Brantom, P. G.; Worrell, N. R.; Sexsmith, C.; Simpson, B. J. (1996): Ninety-day feeding study in Fischer-344 rats of highly refined petroleum-derived food-grade white oils and waxes. In: Toxicologic pathology 24 (2), S. 214–230. DOI: 10.1177/019262339602400210. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Fertility Parameters:
The mean total number of abnormal and normal sperms/findings showed no statistical significances at the end of the treatment period or after the recovery period.
Only the testes weight, weight of the tunica albuginea and the weight of the parenchyma were statistically significantly higher in HD males at the end of the recovery period when compared to control animals.
No histomorphological changes of toxicological concern were observed in testes, epididymides, prostate glands, seminal vesicles, coagulating glands, ovaries, uterus, cervix and vagina.
Thyroid Hormone Measurement:
At the end of the treatment period and after the recovery period, no statistically significant changes were recorded in thyroid hormone levels. - Dose descriptor:
- NOAEL
- Remarks:
- systemic
- Effect level:
- >= 250 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No adverse effects observed at this dose level
- Dose descriptor:
- NOAEL
- Remarks:
- local
- Effect level:
- 40 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No adverse effects observed at this dose level
- Dose descriptor:
- LOAEL
- Remarks:
- local
- Effect level:
- 100 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- Key result
- Critical effects observed:
- no
- Conclusions:
- The test item was tested for subchronic oral toxicity according to OECD TG 408 and in compliance with GLP. The systemic NOAEL was determined to be >= 250 mg/kg bw/day for male and female rats. The local NOAEL was determined to be 40 mg/kg bw/day, based on local irritating/corrosive effects observed in the forestomach of animals treated at 100 and 250 mg/kg bw/day. These effects were a consequence of the test item generating hydrogen chloride as a hydrolysis product in the forestomach.
Table 7: Incidence and Mean Severity of Findings in Gastrointestinal Tract
Dose (mg/kg bw/day) | 0 | 40 | 100 | 250 | ||||
Finding / Groups (Main Test) | ||||||||
Total Animals/Sex Affected / Mean Severity | (10) M | (10) F | (10) M | (10) F | (10) M | (10) F | (10) M | (10) F |
Hyperkeratosis, forestomach | 4/1.0 | 3/1.0 | 2/1.0 | 4/1.0 | 1/1.0 | 2/1.5 | 8/1.0 | 9/1.3 |
Erosion, forestomach | 0 | 0 | 0 | 0 | 0 | 0 | 1/2.0 | 0 |
Ulceration, forestomach | 0 | 0 | 0 | 0 | 0 | 0 | 1/2.0 | 1/1.0 |
Inflammation, forestomach | 0 | 0 | 0 | 0 | 0 | 1/2.0 | 4/1.3 | 9/2.1 |
Hyperplasia, squamous, forestomach | 0 | 0 | 0 | 0 | 0 | 6/1.3 | 7/1.9 | 10/2.3 |
Finding / Groups (Recovery) | ||||||||
Total Animals/Sex Affected / Mean Severity | (5) M | (5) F | - | - | - | - | (5) M | (5) F |
Hyperkeratosis, forestomach | 1/1.0 | 0 | - | - | - | - | 0 | 2/1.0 |
Ulceration, forestomach | 0 | 0 | - | - | - | - | 0 | 1/3.0 |
Inflammation, forestomach | 0 | 0 | - | - | - | - | 0 | 1/2.0 |
Hyperplasia, squamous, forestomach | 1/1.0 | 0 | - | - | - | - | 0 | 2/1.5 |
Table 8: Incidence and Mean Severity of Findings in Lungs
Dose (mg/kg bw/day) | 0 | 40 | 100 | 250 | ||||
Finding / Groups | ||||||||
Total Animals/Sex Affected / Mean Severity | (10) M | (10) F | (0) M | (0) F | (0) M | (1) F | (10) M | (10) F |
Alveolar macrophages | 8/1.9 | 8/1.4 | - | - | - | 0 | 5/1.2 | 5/1.4 |
Inflammation, interstitial | 6/1.7 | 6/1.7 | - | - | - | 0 | 1/3.0 | 1/1.0 |
Pleural inflammation | 0 | 0 | - | - | - | 1/5.0 | 0 | 0 |
Granuloma | 0 | 0 | - | - | - | 0 | 1/1.0 | 0 |
Alveolar/bronchiolar hyperplasia | 1/1.0 | 1/1.0 | - | - | - | 0 | 0 | 1/1.0 |
Table 9: Incidence and Mean Severity of Findings in Mesenteric Lymph Nodes
Dose (mg/kg bw/day) | 0 | 40 | 100 | 250 | ||||
Finding / Groups (Main Test) | ||||||||
Total Animals/Sex Affected / Mean Severity | (10) M | (10) F | (10) M | (10) F | (10) M | (10) F | (10) M | (10) F |
Macrophages, with stored material | 5/1.0 | 9/1.6 | 4/1.8 | 9/1.6 | 7/1.9 | 10/1.8 | 8/2.0 | 10/2.6 |
Inflammation | 0 | 1/1.0 | 0 | 0 | 0 | 1/1.0 | 0 | 0 |
Finding / Groups (Recovery) | ||||||||
Total Animals/Sex Affected / Mean Severity | (5) M | (5) F | - | - | - | - | (5) M | (5) F |
Macrophages, with stored material | 4/1.3 | 4/1.3 | - | - | - | - | 4/1.3 | 5/2.2 |
Inflammation | 0 | 0 | - | - | - | - | 0 | 0 |
Histiocytes foamy | 0 | 0 | - | - | - | - | 0 | 1/3.0 |
Data source
Materials and methods
Results and discussion
Overall reproductive toxicity
- Reproductive effects observed:
- not specified
Applicant's summary and conclusion
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