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EC number: 946-383-6 | CAS number: -
- 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
Repeated dose toxicity: oral
Administrative data
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 21 June 2018 to 13 November 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- OECD testing guideline 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
- Deviations:
- yes
- Remarks:
- See "Any other information" for details
- GLP compliance:
- yes
- Limit test:
- no
Test material
- Reference substance name:
- Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate
- EC Number:
- 946-383-6
- Molecular formula:
- C17H24N2O3
- IUPAC Name:
- Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate
- Test material form:
- liquid: viscous
- Details on test material:
- Name: Trixene AS
Batch no.: OP544-153/1801239973
Appearance: clear, light yellow-green, viscous liquid
Composition: Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate
EINECS-No.: 946-383-6
Molecular formula: C17-H24-N2-O3
Purity: "multi-constituent substance made up of a mixture of isomers
Homogeneity: Homogeneous
Vapour pressure: not stated
Expiry date: 01. Sep. 2019
Storage Room: Temperature (20 ± 5°C)
Constituent 1
- Specific details on test material used for the study:
- No further details specified in the study report.
Test animals
- Species:
- rat
- Strain:
- Sprague-Dawley
- Details on species / strain selection:
- The rat was selected because it is a standard species for use in toxicology studies and is also being used as a rodent species per the current OECD 422 testing guideline.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Animal Information
Species and Strain: Sprague Dawley Rat
Supplier: Charles River Breeding Labs; Raleigh, NC
Method of Identification: Ear tag/cage card
Number of Animals Received: Males: 52; Females: 52
Number Used on Study: Males: 49; Females: 48
Age at First Dose: Males: 11-12 weeks; Females: 11-12 weeks
Weight Range at First Dose: Males: 381.4-467.3g; Females: 245.7-294.7g
Disposition of Extra Animals: Euthanized
Animals were acclimated to laboratory conditions for at least five days prior to the first dose and released from acclimation by a staff veterinarian. During that time, animals were identified by a temporary number that was recorded on each cage label.
Husbandry Information
Feed: Certified Global Teklad Laboratory Diet 2018 (pellets) was provided ad libitum, unless otherwise noted.
Water: Filtered water was provided ad libitum via an automatic watering system, supplemented with water bottles as needed
Bedding: Certified Sani Chips® hardwood bedding
Housing: Animals were housed in one room in polycarbonate cages suspended on stainless steel racks. Each cage was affixed with a cage card containing pertinent animal and study information. Animals were individually housed prior to cohabitation and after confirmation of mating. During cohabitation, one male and one female from the same group were housed together. Dams and pups were group housed.
Temperature Range: 20 to 26°C
Humidity Range: 30 to 70%
Light Cycle: 12-hour light/12-hour dark, interrupted as necessary for study-related events
Air Changes: Minimum of 10 air changes per hour
The feed was analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The water is routinely analyzed for contaminants and specific microbes. The bedding was analyzed by the manufacturer for acceptable levels of heavy metals, aflatoxins, bacteria, yeasts, molds, and organophosphates prior to certification. No contaminants were known to be present in the feed, water, or bedding at levels that might have interfered with achieving the objectives of the study.
Actual temperature and relative humidity in the animal room were monitored continuously by a computerized system. All environmental parameters were maintained within the protocol requirements.
In addition to standard husbandry procedures, environmental enrichment was provided according to company SOPs.
Administration / exposure
- Route of administration:
- oral: gavage
- Details on route of administration:
- The oral route was selected because it is the relevant route of exposure to humans and is also being used per the current OECD 422 testing guideline.
The dose levels were selected based on the results from previous toxicology studies performed by the Sponsor at Smithers Avanza (Study # 2386-14351). - Vehicle:
- peanut oil
- Details on oral exposure:
- Dose Formulations
The neat test and vehicle/control substance were considered 100% pure for formulation purposes.
The vehicle/control substance, peanut oil, was used as received; no formulations were necessary.
Formulations were prepared four times over the course of the study and assigned a 28-day shelf life. Group 4 (500 mg/mL) formulations were prepared by adding the appropriate amount of test substance to the required amount of peanut oil, then vortexed to achieve uniformity and inverted approximately 30 times. Formulations for Groups 3 (150 mg/mL) and 2 (50 mg/mL) were prepared by serially diluting the Group 4 formulation with the required amount of peanut oil until the required final volume was reached. Formulations were vortexed to achieve uniformity and inverted approximately 30 times. All formulations were stored refrigerated (5±3°C) until used for dosing.
Dose Administration
Formulations were placed at room temperature prior to dosing. Dosing volumes were based on the animals’ most recent body weights. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Stability and homogeneity analysis of the dose formulations was performed under Smithers Avanza Study No. 2386-14350 as part of the method validation.
Samples were analyzed for concentration verification by Smithers Avanza using a validated method. The analytical methods are described in the Dose Formulation Analysis Report. - Duration of treatment / exposure:
- F0 males were dosed for 35 days. Animal 27455 (4m) that replaced Animal 27116 (4m) on SD 8 received 27 doses.
The F0 females were dosed for two weeks prior to cohabitation, during cohabitation, through gestation, and to at least postnatal day (PND) 12. The first day of dosing was designated as SD 1 for each animal. - Frequency of treatment:
- The animals were dosed daily via oral gavage at a volume of 2 mL/kg.
Doses / concentrationsopen allclose all
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Peanut oil
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 24 animals per dose group (12 male/12 female)
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- Group Assignment and Doses
Animals were initially accepted into the randomization pool based upon prestudy body weights, physical examinations,. They were assigned to study groups using computer-generated random numbers such that the mean body weight for each group was not statistically different (p ≤ 0.05) from the control mean. Males and females were randomized separately. Following randomization each study animal was assigned a unique number. - Positive control:
- Not required.
Examinations
- Observations and examinations performed and frequency:
- Observations
Cageside observations included observation for mortality, moribundity, general health, and signs of toxicity. Physical examinations included evaluation of skin and fur characteristics, eye and mucous membranes, respiratory, circulatory, autonomic, and central nervous systems, and somatomotor and behavior patterns. See "Any other information" for further details.
For the FOB, animals were transported to the testing room and acclimated to white noise for at least 10 minutes prior to testing. See "Any other information" for further details.
Clinical Pathology
Blood samples were collected from five selected animals/sex/group. Animals were fasted overnight (with water available) prior to sample collection on SD 35 (males), and PND 13 (parturient females). See "Any other information" for further details.
Hormone Analysis
Blood Collection from F0 Generation
On the day of necropsy, blood was collected from animals via cardiac puncture under 70% CO2/ 30% O2 inhalant anesthesia. Animals were fasted for sample collection. At least 0.6 mL was collected into serum separator tubes. Immediately following blood collection, animals were euthanized via carbon dioxide asphyxiation followed by necropsy. - Sacrifice and pathology:
- Termination
Moribund animals were euthanized by carbon dioxide inhalation followed by exsanguination prior to necropsy.
Nonparturient females not delivering by SD 57 were euthanized by carbon dioxide asphyxiation and exsanguination and necropsied (uterus and ovaries were examined).
On SD 36 for F0 males, PND 13 or 14 for F0 females, all surviving animals were euthanized by carbon dioxide inhalation followed by exsanguination prior to necropsy.
Necropsy
Animals were fasted overnight (with water available) prior to necropsy. Animals were necropsied as soon as possible after the time of death or discovery. Animals were necropsied, bone marrow smears were prepared (five F0 animals/sex/group at scheduled necropsy), required organs were weighed, and protocol-specified tissues were collected and preserved. No organ weights were collected from animals found dead or euthanized prior to scheduled termination and no bone marrow smears were prepared from animals found dead.
Gross necropsy of F0 animals included examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents. All reproductive organs (testes, epididymides, prostate, seminal vesicles with coagulating glands, ovaries, uterus with cervix; sex appropriate) were collected and weighed as soon as possible after dissection; paired organs were weighed together. For five selected animals/sex/group at scheduled necropsy, additional protocol-specified tissues were collected and weighed as soon as possible after dissection; paired organs were weighed together. The following organs were collected/weighed as follows: thyroid with parathyroids, seminal vesicle with coagulating gland, and uterus with cervix.
Tissues were preserved in 10% neutral buffered formalin (NBF) with the exception of the eyes (and associated ocular tissue) and testes (with epididymides), which were preserved in modified Davidson’s fixative and subsequently transferred to 10% NBF. Tissues from animals found dead were preserved in 10% NBF only. Two bone marrow smears were prepared from the left femur and the slides were air-dried, fixed in methanol, and stored at room temperature for possible future evaluation. No analysis of the bone marrow smears was deemed necessary; therefore, the unstained slides were discarded prior to report finalization.
Histopathology
Preserved tissues from the five females and males selected in the control and high dose study groups were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically by a board-certified veterinary pathologist. The testis was also stained with periodic acid-Schiff stain (PAS-H) and examined microscopically by a board-certified veterinary pathologist with special emphasis on stages of spermatogenesis and interstitial testicular cell structure.
The spleen in both sexes and the testes and cecum in males were identified as potential target tissues by the pathologist, therefore these tissues from selected animals in Groups 2 and 3 were processed to slides and microscopically examined by the pathologist. - Other examinations:
- Other examinations with regards the reproductive/developmental part of the study are detailed in Section 7.8.1.
- Statistics:
- Group mean, standard deviation, and sample size are reported for all quantitative measurement data.
Quantitative data (body weights, body weight changes, food consumption, clinical pathology, and organ weight data) from the treated groups were compared statistically to the data of the control group using one-way Analysis of Variance (ANOVA) techniques; sexes were analyzed separately. A Shapiro-Wilk test was used to test for normality, followed by the Levene’s test to test the hypothesis of homogeneity of variances. If either the normality or homogeneity test failed (as indicated by a Shapiro-Wilk or Levene’s test p-value ≤ 0.01), the data were transformed using log-transformed values and both the Shapiro-Wilk and Levene’s tests were repeated with the log-transformed values. If the results from either the Shapiro-Wilk or Levene’s test on the log-transformed data failed, the analysis of the data continued using rank-transformed data using Kruskal-Wallis ANOVA. If both the Shapiro-Wilk or Levene’s test were not statistically significant, the ANOVA was performed on the untransformed or log-transformed data, respectively. The Dunnett’s t-test was used to determine which groups (if any) differed from the control group. Group comparisons were evaluated at the 0.05 (two-tailed) probability level. An arcsine square root transformation was used for some proportion/percentage data which do not meet the assumptions of parametric statistical tests in the attempt to normalize the data; this data was transformed prior to the ANOVA analysis. The term “significant” is used throughout the text of the report to indicate statistical significance at p ≤ 0.05.
Dunnett’s t-test was used to determine if differences exist between the control and treated group(s). Group comparisons were evaluated at the 5% two-tailed probability level.
Results and discussion
Results of examinations
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- Treatment with Trixene AS did not affect physical examinations or cageside observations. Observations noted during physical examinations were considered incidental and not related to test substance due to lack of an associated dose response. The following abnormalities were observed in the males at various study intervals: alopecia of the forepaws (1m 27053) or hindlimbs/forepaw digits (4m-27117), hunched posture (4m-27117), and abrasion of the neck (3m 27097; 300 mg/kg/day).
The following observations were observed in females at various study intervals: alopecia on the forepaw digits (2f-27090; 3f-27107) or forelimbs (2f-27083) abrasion/alopecia of the abdomen (1f-27061), and alopecia of the chest (3f-27114). No abnormalities were observed during cageside observations. - Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- Treatment with Trixene AS did not affect mortality. There was one male and three females that died during the study, but all deaths were unrelated to the test substance. Animal 27116 (4m; 1000 mg/kg/day) had a rough haircoat and was vocalizing continuously on SD 8. This animal also had body weight loss and low food consumption. The attending veterinarian examined the animal and treated with a diet gel recovery pack. The animal was euthanized as moribund on SD 8 and replaced with Animal 27455. The death was not considered test substance related due to the low incidence morbidity with observations (low body weight and food consumption) that were not observed in other animals. Additionally, all other males survived until the scheduled termination.
Females 27063 (1f; 0 mg/kg/day) and 27064 (1f, 0 mg/kg/day) were pale with a rough haircoat on GD 27, and Female 27064 was also severely languid on GD 23. Female 27063 (1f) had suspected delayed parturition and after examination by the attending veterinarian, both animals were euthanized as moribund on the respective gestational days. Animal 27131 (4f; 1000 mg/kg/day) was found dead on GD 23. Red discharge from the vagina was noted at necropsy in all three animals, however the cause of death was undetermined based on microscopic evaluation of limited tissues by the pathologist. All of the unscheduled deaths were considered unrelated to the test substance and they were suspected to be a complication with pregnancy, as they occurred during late gestation and occurred in the control group and at low incidence in Group 4 females. All other females survived until the scheduled termination. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Treatment with Trixene AS impacted body weights and body weight changes of males. Mean body weights of Groups 2 (100 mg/kg/day) and 4 (1000 mg/kg/day) were significantly lower than the control from SD 15 to 35. Mean body weights of Group 3 (300 mg/kg/day) were significantly lower on SD 29 and 35. Mean body weight changes of treated groups were significantly lower than the control at various study intervals, and all groups had significantly lower absolute body weight change from SD 1 to 35. Although, males in all groups had a significantly lower body weight change, all groups gained weight and there was no clear dose response; therefore the effect on body weight was not considered adverse.
Treatment with Trixene AS impacted body weights of females. Mean body weight of females were significantly lower than the control at the following intervals: SD 15 2f, GD 0–2f, 3f; GD 7–2f, 3f; GD 14–all; GD 20 – 3f; PND 0–all; PND 4–all, PND 7–4f. Treated groups often had significantly lower mean body weight change compared to the controls. Mean absolute body weight change from SD 1 to 15 was significantly lower in all treated groups compared to the control. Mean body weight change from PND 4 to 7 was significantly higher in all treated groups Although, females in all groups had variable body weight changes compared to the controls at varying intervals, all groups gained weight and there was no clear dose response; therefore the effect on body weight was not considered adverse. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Treatment with Trixene AS impacted food consumption. Mean total food consumption (SD 1-15) of treated groups was lower compared to the control, with significant differences for Groups 2 (100 mg/kg/day) and males in Group 4 (1000 mg/kg/day). Females in Groups 3 and 4 from SD 1-8 and females in Group 2 from SD 8- 15 had significantly lower mean food consumption compared to the control. Mean food consumption of females in Group 4 was also significantly lower than the control from PND 7-12. Although, there were variable effects on food consumption and body weight changes throughout the study, all animals gained weight. Thus, the effects on food consumption were not considered adverse.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Males (Study Day 36)
Test substance-related decreases in measures of erythroid mass (red blood cell count, hemoglobin concentration and hematocrit percent) with concurrent increases in reticulocyte counts occurred in males at all doses. In males administered 1000 mg/kg/day, decreases in erythroid parameters ranged from 12 14% and reticulocyte counts were 47% increased. In males administered 300 mg/kg/day, decreases in erythroid parameters ranged from 9-11% and reticulocyte counts were 14% increased. In males administered 100 mg/kg/day, decreases in erythroid parameters ranged from 6-7% and reticulocyte counts were 12% increased. No correlating changes in bone marrow morphology were observed microscopically; however, minimally increased erythropoiesis was observed in the spleen of 4 of 5 males administered 1000 mg/kg/day. Decreases in measures of erythroid mass with concurrent increases in reticulocyte count were suggestive of blood loss and/or erythrocyte injury with a normal regenerative response.
Test substance-related changes in white blood cell counts consisted of a mild (66%) increase in lymphocyte counts at a dose level of 1000 mg/kg/day. Although increases in lymphocyte counts were observed at dose levels of 100 or 300 mg/kg/day, the magnitudes of the increases were within normal variation and could not be attributed definitively to the test substance. Decreases in eosinophil counts at all doses were considered equivocal due to the lack of a dose-response; however, would be consistent with stress in a rodent.
No test substance-related changes in coagulation parameters were observed.
Females (Post Natal Day 13)
Test substance-related decreases in erythroid parameters (red blood cell count, hemoglobin concentration and hematocrit percent) with concurrent increases in reticulocyte counts occurred in females at ≥300 mg/kg/day. In females administered 1000 mg/kg/day, decreases in erythroid parameters ranged from 7-9% and reticulocyte counts were 45% increased. In females administered 300 mg/kg/day, decreases in erythroid parameters ranged from 5-6% and reticulocyte counts were 29% increased. No correlating changes in bone marrow morphology were observed microscopically; however, mildly increased erythropoiesis was observed in the spleen of one female administered 1000 mg/kg/day. Decreases in measures of erythroid mass with concurrent increases in reticulocyte count were suggestive of blood loss and/or erythrocyte injury with a normal regenerative response.
No test substance-related changes in coagulation parameters were observed - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Males (Study Day 36)
Test substance-related changes in clinical chemistry consisted of nearly 2-fold increases in alanine transaminase activities in males administered 1000 mg/kg/day that were consistent with minimal hepatocellular injury; however, no correlating microscopic observations occurred in the liver, therefore this was not considered an adverse effect.
Females (Post Natal Day 13)
Test substance-related changes in clinical chemistry consisted of 2-fold increases in alanine transaminase in females administered 1000 mg/kg/day that were consistent with minimal hepatocellular injury and approximately 2-fold increases in alkaline phosphatase activities in females administered ≥300 mg/kg/day. No correlating microscopic observations occurred in the liver. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- For functional observational battery parameters exopthalmus and palpebral closure, observations were recorded for each eye, but findings are presented in the data for both eyes.
Treatment with Trixene AS had no effect on the functional observation battery. A score of zero was considered normal. During handheld observations, Animal 27068 (2m) had no reactivity to handling (score of 2) and Animal 27137 (4f) exhibited salivation (score of 1). Animal 27121 (4m) exhibited low activity during the open field observations (score of 1). These observations were not considered test substance-related due to the low incidence. Means of quantitative measures (grip strength, hindlimb splay, grooms, fecal boli, urine pools, and rears) were comparable across groups. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Males (Study Day 36)
Test substance-related increases in group mean spleen weight parameters were observed in males at doses ≥ 300 mg/kg/day. Increases in spleen weight parameters in males administered 300 mg/kg/day were equivocal. Increases in spleen weight parameters in males administered 1000 mg/kg/day correlated with microscopic increased splenic erythropoiesis.
Non dose-related minimal decreases in thymus weight parameters were observed at all doses in males. Given the lack of a dose-response, these changes could not be attributed to the test substance; however, they were consistent with stress in a rodent and correlated with decreased eosinophil counts.
Females (Post Natal Day 13)
No test substance-related changes in group mean organ weight parameters were observed in females on PND 13 or 14; however, minimal test substance-related increases in spleen weight parameters occurred in one female administered 1000 mg/kg/day that correlated with microscopic increased splenic erythropoiesis. - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No test substance-related macroscopic observations occurred in males on SD 36 or females on PND 13 or 14.
All gross alterations seen in the current study were typical of sporadic, naturally occurring background changes commonly observed in rats of this age and strain. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Males (Study Day 36)
Test substance-related changes occurred in the spleen and the testes. In the spleen, minimally increased erythropoiesis was observed in the red pulp of 4 of 5 males administered 1000 mg/kg/day and correlated with increased spleen weight parameters. In the testes of 3 of 5 males administered 1000 mg/kg/day, spermatid retention was characterized by spermatozoa at the luminal surface of some, but not all, Stage IX tubules. Notably, spermiation occurred normally within most tubules. In affected tubules there were also a few spermatozoa basally located within Sertoli cell cytoplasm oriented parallel to the basement membrane.
A finding of uncertain relationship to test substance administration consisted of minimal to mild mucosal regeneration with minimal to mild lymphocytic infiltration of the lamina propria in the cecum of males at all doses. Two of 5 males administered 100 mg/kg/day, 4 of 5 males administered 300 mg/kg/day and 3 of 5 males administered 1000 mg/kg/day were affected. These changes were characterized by the expansion of the lamina propria by a cell population of primarily lymphocytes and macrophages with regeneration of the overlying mucosa characterized by increased basophilia of the epithelium. Given the lack of similar changes in females, the lack of correlating clinical observations, and the lack of a clear dose response, the relationship of this change to the test substance was uncertain.
Females (Post Natal Day 13)
Test substance-related changes occurred in the spleen at doses ≥300 mg/kg/day. In the spleen, mildly increased erythropoiesis was observed in the red pulp of 1 of 5 females administered 1000 mg/kg/day and correlated with increased spleen weight parameters in that individual (27135). Additionally, 3 of 5 females administered 300 mg/kg/day had minimally increased erythropoiesis in the spleen.
Notably, one female administered 300 mg/kg/day had mildly increased hematopoiesis in the spleen. This was not considered test substance-related, as this individual also had a cutaneous ulcer that was considered the likely cause for the increased hematopoiesis in the spleen.
All other microscopic alterations seen in the current study were typical of sporadic, naturally occurring background changes commonly observed in rats of this age and strain. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- Hormone Levels
Treatment with Trixene AS affected thyroxine (T4) levels of males. Mean T4 levels of treated groups were significantly lower than the control group in a dose-dependent manner. Although there was a test-substance related decrease in T4 levels, there was no associated effects on thyroid organ weight and histopathological effects, therefore this was not considered an adverse effect. - Details on results:
- Mortality was not effected by administration of the test substance. Two control females and one male and female administered 1000 mg/kg/day were found dead or euthanized in a moribund condition. The cause of death was undetermined based on gross observations and limited microscopic tissue evaluation.However, the deaths were not considered test substance-related because the deaths in the females were considered to be complications of parturition and because of the low incidence of deaths in males.
Test substance-related changes in body weight and food consumption were observed but were not considered adverse. Body weight and food consumption were statistically significantly decreased compared to the control. All groups gained weight and there was no clear dose response; therefore the effect on body weight was not considered adverse.
Test substance-related changes in thyroid hormone levels of F0 male rats were observed. Mean T4 levels of treated groups were significantly lower than the control group in a dose-dependent manner. In the absence of thyroid organ weight and histopathological effects this finding was not considered adverse. Furthermore, no other toxicologically significant observations with respect to survival, clinical observations, body weights, food consumption, and ophthalmology were observed.
Test substance-related changes in clinical pathology parameters were limited to ~2-fold increases in ALTi activities in animals administered 1000 mg/kg/day consistent with minimal hepatocellular injury; and decreases in measures of erythroid mass with increases in reticulocyte counts in males at all doses and females administered ≥300 mg/kg/day consistent with blood loss or erythrocyte injury and a normal erythrocytic regenerative response. No correlating microscopic changes occurred in the liver; however, test substance-related increased erythropoiesis was observed in the spleen of 4 of 5 males and 1 of 5 females administered 1000 mg/kg/day and 3 of 5 females administered 300 mg/kg/day which correlated with the increases in reticulocyte counts. None of these changes were considered adverse, as the effects on erythroid mass were not of a magnitude to affect the overall well-being of the animal.
Effect levels
open allclose all
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 300 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- gross pathology
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- gross pathology
Target system / organ toxicity
- Key result
- Critical effects observed:
- no
- Lowest effective dose / conc.:
- 300 mg/kg bw/day (nominal)
- System:
- male reproductive system
- Organ:
- cauda epididymis
Applicant's summary and conclusion
- Conclusions:
- This study determined the potential toxicity of Trixene AS when administered daily for at least 28 days via oral gavage to male and female Sprague Dawley rats and the potential reproductive and developmental toxicity.
Mortality was not effected by administration of the test substance. Two control females and one male and female administered 1000 mg/kg/day were found dead or euthanized in a moribund condition. The cause of death was undetermined based on gross observations and limited microscopic tissue evaluation. However, the deaths were not considered test substance-related because the deaths in the females were considered to be complications of parturition and because of the low incidence of deaths in males.
Test substance-related changes in body weight and food consumption were observed but were not considered adverse. Body weight and food consumption were statistically significantly decreased compared to the control. All groups gained weight and there was no clear dose response; therefore the effect on body weight was not considered adverse.
Test substance-related changes in thyroid hormone levels of F0 male rats were observed. Mean T4 levels of treated groups were significantly lower than the control group in a dose-dependent manner. In the absence of thyroid organ weight and histopathological effects this finding was not considered adverse. Furthermore, no other toxicologically significant observations with respect to survival, clinical observations, body weights, food consumption, and ophthalmology were observed.
Test substance-related changes in clinical pathology parameters were limited to ~2-fold increases in ALTi activities in animals administered 1000 mg/kg/day consistent with minimal hepatocellular injury; and decreases in measures of erythroid mass with increases in reticulocyte counts in males at all doses and females administered ≥300 mg/kg/day consistent with blood loss or erythrocyte injury and a normal erythrocytic regenerative response. No correlating microscopic changes occurred in the liver; however, test substance-related increased erythropoiesis was observed in the spleen of 4 of 5 males and 1 of 5 females administered 1000 mg/kg/day and 3 of 5 females administered 300 mg/kg/day which correlated with the increases in reticulocyte counts. None of these changes were considered adverse, as the effects on erythroid mass were not of a magnitude to affect the overall well-being of the animal.
The only other test substance-related microscopic change occurred in the testes of 3 of 5 males administered 1000 mg/kg/day and consisted of spermatid retention in some, but not all, Stage IX tubules. Notably, spermiation occurred normally within most tubules and no increase in cellular debris or hypospermia was observed in the epididymides of affected animals. Spermatid retention was considered an adverse change due to the potential effects on reproduction.
In summary, Trixene AS induced repeat dose toxicity in male and female rats. The repeat dose no-observed-adverse-effect level (NOAEL) for male rats was determined to be 300 mg/kg/day due to microscopic findings (spermatid retention). The repeat dose NOAEL for female rats was considered to be 1000 mg/kg/day, the highest dose level tested. - Executive summary:
Trixene AS: Combined Oral Gavage Repeated Dose Toxicity Study Including Reproduction/Developmental Toxicity Screening in Male and Female Sprague Dawley Rats
The purpose of this study was to determine the potential toxicity of Trixene AS when administered once daily for at least 28 days via oral gavage to male and female Sprague Dawley rats and to determine the potential reproductive and developmental toxicity.
Ninety-six (48/sex) Sprague Dawley rats were randomly assigned to four groups (12 animals/sex). Animals were administered control substance (Peanut Oil) or Trixene AS at 100, 300, or 1000 mg/kg once daily via oral gavage for at least 28 days. Females were dosed for two weeks prior to cohabitation, during cohabitation, through gestation, and to at least postnatal day (PND) 12. Animals were subjected to a full gross necropsy on Study Day (SD) 36 (F0 males), or PND 13 or 14 (parturient F0 females and pups). Females that did not litter were subjected to a full gross necropsy on SD 57.
Parameters evaluated during the study for the F0 generation included mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, functional observation battery, vaginal cytology, clinical pathology (clinical chemistry, hematology, and coagulation), thyroid hormone (T4) analysis (males only), gross pathology findings, absolute and relative organ weights, and histopathology findings.
Treatment with Trixene AS at doses ≥300 mg/kg/day had no effect on the parameters listed above. Test substance-related changes in body weight and food consumption were observed but were not considered adverse at doses ≥100 mg/kg/day. Body weight and food consumption were statistically significantly decreased compared to the control. All groups gained weight and there was no clear dose response; therefore, the effect on body weight and food consumption were not considered adverse.
Test substance-related changes in thyroid hormone levels of F0 male rats were observed. Mean T4 levels of treated groups were significantly lower than the control group in a dose-dependent manner. In the absence of thyroid organ weight and histopathological effects this finding was not considered an adverse response. Furthermore, no other toxicologically significant observations with respect to survival, clinical observations, body weights, food consumption, and ophthalmology were observed.
Test substance-related changes in clinical pathology parameters were limited to ~2-fold increases in ALTi activities in animals administered 1000 mg/kg/day consistent with minimal hepatocellular injury; and decreases in measures of erythroid mass with increases in reticulocyte counts in males at all doses and females administered ≥300 mg/kg/day consistent with blood loss or erythrocyte injury and a normal erythrocytic regenerative response. No correlating microscopic changes occurred in the liver; however, test substance-related increased erythropoiesis was observed in the spleen of 4 of 5 males and 1 of 5 females administered 1000 mg/kg/day and 3 of 5 females administered 300 mg/kg/day which correlated with the increases in reticulocyte counts. None of these changes were considered adverse, as the effects on erythroid mass were not of a magnitude to affect the overall well-being of the animal.
The only other test substance-related microscopic change occurred in the testes of 3 of 5 males administered 1000 mg/kg/day and consisted of spermatid retention in some, but not all, Stage IX tubules. Notably, spermiation occurred normally within most tubules and no increase in cellular debris or hypospermia was observed in the epididymides of affected animals. Spermatid retention was considered an adverse change due to the potential effects on reproduction.
In summary, Trixene AS induced repeat dose toxicity in male and female rats at >300 mg/kg/day. The repeat dose no-observed-adverse-effect level (NOAEL) for male rats was determined to be 300 mg/kg/day due to microscopic findings (spermatid retention). The repeat dose NOAEL for female rats was considered to be 1000 mg/kg/day, the highest dose level tested.
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